Immunization agents and methods of use

ABSTRACT

The present invention relates to a vaccine composition comprising an effective amount of a replication-competent controlled recombinant virus. Further encompassed are uses in immunization and methods of immunization employing compositions comprising a replication-competent controlled recombinant virus of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/530,715, filed Feb. 21, 2017, which is the national stage ofinternational application No. PCT/EP2015/069472, filed Aug. 25, 2015,which claims the benefit of U.S. Provisional Application Nos.62/070,443, filed 26 Aug. 2014; and 62/122,088, filed 10 Oct. 2014, bothof which are incorporated herein by reference in their entirety;including any drawings.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“VIR2A-PCT_ST25.txt”, created 25 Aug. 2015, which is 4 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to certain replication-competentcontrolled viruses and their utilization for immunization.

BACKGROUND OF THE INVENTION

Vaccination is most probably the most cost-effective medicalintervention that has saved countless human lives during its history ofmore than two hundred years. Among its most spectacular successes countthe eradication of smallpox as well as the virtual disappearance ofdiphtheria, tetanus and paralytic poliomyelitis. Andre, F. E. (2003)Vaccinology: past achievements, present roadblocks and future promises.Vaccine 21: 593-5. In addition, vaccination has controlled, in at leastpart of the world, yellow fever, pertussis, Haemophilus influenzae typeb, measles, mumps, rubella, typhoid and rabies. Still, importantinfections remain unpreventable as well as incapable of being treated bya therapeutic vaccine. Moreover, effectiveness of a number of vaccinesis less than would be desirable. Clearly, the creation of new vaccineshas not become routine, and development of immunization agents againstcertain diseases may require new approaches. The following examplesillustrate some of the current challenges.

About 50% of ten-year-old American and European children areseropositive for herpes simplex virus HSV-1. Stanberry, L. R. Herpessimplex virus vaccines. In: Vaccines (Plotkin, S. A. et al., eds.) 5thedition. 2008. Saunders Elsevier. Prevalence increases to 70-80% in thesixth and seventh decades. Herpes simplex virus HSV-2 prevalence risesduring adolescence and reaches peaks of 20% and 30% in Europe and theUnited States, respectively. Potentially life-threatening infection canoccur in subjects with skin disorders. HSV infection of the eye mayaffect the conjunctiva, cornea or retina and can cause blindness.Perinatal herpes infection can include encephalitis or disseminatedinfection. Infection in immune-compromised subjects such as HIV patientsmay also be life-threatening. HSV infection has many complications,resulting in significant morbidity and mortality. It is noteworthy thatacute genital herpes infection significantly increases the risk of HIVacquisition. HSV-1 and HSV-2 infections involve replication at the siteof entry and spread of virus along nerve fibers to sensory ganglia wherelatency is established. The viruses can reactivateperiodically/sporadically, migrate back to the periphery and replicatein the periphery. A number of prophylactic and therapeutic vaccinecandidates were developed and tested clinically. Stanberry, L. R.(2008). At this time, no effective therapeutic or prophylactic vaccineis available. It has been argued that a successful therapeutic orprophylactic vaccine should elicit powerful antibody as well as T cell(Th-1) responses. Cunningham, A. L. and Mikloska, Z. (2001) The holygrail: immune control of human herpes simplex virus infection anddisease. Herpes 8 (Supplement 1): 6A-10A.

It has been suggested that the goal of developing a prophylactic HSV-1or HSV-2 vaccine may be too ambitious and that the focus should be ongenerating a therapeutic vaccine. In this regard, it is encouraging thata vaccine was able to be developed that shows effectiveness inpreventing shingles (herpes zoster). Johnson, R. et al. (2007)Prevention of herpes zoster and its painful and debilitatingconsequences. Int. J. Infect. Dis. 11 (Supplement 2): S43-S48. Thelatter disease is caused by reactivation in sensory ganglia ofvaricella-zoster virus (VZV), another alphaherpes virus. The vaccine,made from the live attenuated Oka strain, is >60% effective in reducingthe burden of illness or postherpetic neuralgia. This protection isassociated with a boosted cell-mediated immune response. The Oka strainwas also utilized to develop a highly effective, live attenuated vaccinefor the prevention of chicken pox/varicella. Gershon, A. A. et al.Varicella vaccine. In: Vaccines (Plotkin, S. A. et al., eds.) 5thedition. 2008. Saunders Elsevier. Therefore, it can be argued that itshould not be impossible to create effective herpes simplex vaccines. Itis noted that development of an even more effective herpes zostervaccine (or a varicella vaccine that cannot reactivate) would be aworthwhile goal.

Influenza is characterized typically by sudden fever, sore throat,cough, headache, myalgia, chills, anorexia and fatigue. Bridges, C. B.et al. Inactivated influenza vaccines. In: Vaccines (Plotkin, S. A. etal., eds.) 5th edition. 2008. Saunders Elsevier; Belshe, R. B. et al.Influenza vaccine-live. In: Vaccines (Plotkin, S. A. et al., eds.) 5thedition. 2008. Saunders Elsevier. Influenza is a high morbidity butrelatively low mortality disease. Seasonal attack rates typically arebetween 5% and 20%. The death toll from complications of the illness isconsiderable. According to the WHO, the worldwide yearly death toll maylie between 250,000 and 500′000. Influenza viruses are enveloped andcontain a segmented negative-sense RNA genome. The spherical viralparticles have spikes consisting of hemagglutinin (HA) and neuraminidase(NA). HA is the major antigen against which the host antibody responseis directed. Influenza A viruses are classified into subgroups based onthe properties of their envelope proteins HA and NA. Sixteen H(A) andnine N(A) subtypes are currently known. Presently, influenza viruses ofsubtypes H1N1, H1N2 and H3N2 are circulating in humans. Influenza type Aalso infects birds including poultry, pigs, horses, dogs and even seamammals. All known HA and NA subtypes could be isolated from wildaquatic birds, which constitute a natural reservoir and a source ofgenes for pandemic A-type viruses. Because of the error-prone mode ofreplication and selection in the host, influenza A and B viruses undergogradual antigenic change in their two surface antigens, the HA and NAproteins. This phenomenon known as antigenic drift necessitatescontinuous vigilance and yearly review/update of strains used forvaccine production. Pandemics result from antigenic shift, i.e.,introduction into the human population of a novel influenza A viruscontaining either only a novel HA subtype or both novel HA and NAsubtypes.

Whole-virus inactivated influenza vaccines have been in use since 1945.Typically, vaccine viruses have been propagated in the allantoiccavities of embryonated hens' eggs. More recently, such vaccines alsohave been made from viruses amplified in mammalian cell lines. Since the1970s, most inactivated vaccines are subvirus or split vaccines. Typicalvaccines in use are trivalent, comprising HAs from H1N1 and H3N2 subtypeinfluenza A strains and an influenza B strain (referred to as TIV). Thevariable efficacy of the inactivated virus vaccines (TIV), the shortduration of protection, adverse reaction to parenteral administration(the primary route used) and the absence of induction of effectivecellular immunity has led to the development of live attenuatedinfluenza virus vaccines (LAIV). An intranasal vaccine was made based ontemperature-sensitive and cold-adapted influenza virus A and B strains.

An updated systematic review and meta-analysis of vaccine efficacy andeffectiveness data was published not long ago. Osterholm, M. T. et al.(2012) Efficacy and effectiveness of influenza vaccines: a systematicreview and meta-analysis. Lancet Infect. Dis. 12: 36-44. The analysisfocused on studies carried out in the United States and publishedbetween 1967 and 2011. Studies were selected based on a set of criteriathat were intended to ensure scientific rigor and, to the extentpossible, exclude bias. All criteria were fulfilled by 17 randomized,controlled trials showing vaccine efficacy (95% CI>0), of which trials 8related to TIV and 9 to LAIV. The trials covered 24 influenza seasonsand included almost 54,000 participants. Of the trials that revealedsignificant efficacy for TIV, 6 involved 18-64-year-old participants,one children aged 6-23 months and one included all age groups andreported a combined efficacy. The mean vaccine efficacy revealed bythese trials was 62%. It is noted that none of the trials specificallytested vaccine effects in adults 65 years of age and older or inchildren aged 2-17. Of particular interest is a study on young childrenthat was carried on over two seasons in both of which there was a goodmatch between vaccine and circulating strains. Hoberman, A. et al.(2003) Effectiveness of inactivated influenza vaccine in preventingacute otitis media in young children: a randomized controlled trial.JAMA 290: 1608-16. Vaccine efficacy in the first season was 66% and inthe second −7%. Regarding LAIV, mean efficacy from eight studies inchildren aged 6 months to 7 years was 78%. Osterholm, M. T. et al.(2012). Three studies on subjects aged 18-49 revealed no significantprotection. One study in persons over 60 showed an overall efficacy of42%, but efficacy in 60-69-year-olds seemed to be considerably lowerthan that in persons over 70. No qualifying study related to childrenaged 8-17 or adults between 50 and 59 years of age.

Nine of 14 observational studies that satisfied the inclusion criteriareported effectiveness of seasonal influenza vaccine. Osterholm, M. T.et al. (2012). These studies included 17 embedded or cohort analyses.Six of the 17 analyses (35%) showed significant effectiveness againstmedically attended, laboratory-confirmed influenza. In children of 6-59months, significant vaccine effectiveness was found in 3 of 8 seasons(38%). One of two such studies reported vaccine effectiveness insubjects aged 65 and older. Based on these data it can be concluded thatcurrently available influenza vaccines provide moderate protectionagainst virologically confirmed disease, which protection is notlong-lasting. No protection may be obtained in some seasons. Evidencefor protection of the highest risk population, i.e., persons 65 years ofage or over, is very thin indeed. More effective vaccines are clearlyneeded. Present vaccines rely largely on the induction of HA antibodiesfor protective effects. It has been proposed that future influenzavaccines should be capable of inducing potent (effector) T cellresponses, i.e., should induce more complete immune responses.Osterhaus, A. et al. (2011) Towards universal influenza vaccines. Phil.Trans. R. Soc. B 366: 2766-73; Thomas, P. G. et al. (2006) Cell-mediatedprotection in influenza infection. Emerging Infectious Diseases 12:48-54.

HIV/AIDS is a leading cause of death in Subsaharan Africa and animportant cause of mortality worldwide. HIV are lentiviruses which areretroviruses that cause characteristically slow infections, producingdisease after long latency periods in the presence of an activated hostimmune response. HIV includes HIV-1 and HIV-2, with HIV-1 being the moreaggressive and more rapidly spreading virus. As a first step of theinfection process, viral envelope protein gp120 binds to CD4 receptorand then to CCR-5 or CXCR-4 co-receptors on the surface of target cells.CD4 is present in T helper lymphocytes, monocytes-macrophages,follicular DC, Langerhans cells in the skin and microglia in the centralnervous system.

At this time, there is no effective vaccine available against HIV/AIDS.A number of observations suggest that, in order to be effective, avaccine must trigger a substantial cellular immune response. A number ofvaccine candidates were tested in clinical trials. Recent pivotal trialsmade use of viral vectors to induce T-cell responses. To furtherincrease efficacy, these trials also implemented prime-boost regimens.One such phase III trial used a combination of a priming canarypox virusexpressing an HIV gp120 antigen and an HIV gp120 boost. Although a trendtowards prevention of HIV-1 was found, the vaccine produced nobeneficial effects on post-infection virus load or CD4+ cell counts.Draper, S. J. and Heeney, J. L. (2010) Viruses as vaccine vectors forinfectious diseases and cancer. Nat. Rev. Microbiology 8: 62-73; Kim, J.H. et al. (2012) HIV vaccines—lessions learned and the way forward.Curr. Opin. HIV AIDS 5: 428-34. Another recent series of trials usingreplication-incompetent Ad5 expressing various HIV proteins as vaccinesdid not reveal any indication of efficacy. The latter experiences aswell as the realization that a narrow CTL response can lead to theappearance of CTL escape mutants suggest that future vaccine candidatesshould be capable of inducing complete immune responses including broadCTL responses. Goulder, P. J. R. and Watkins, D. I. (2004) HIV and SIVCTL escape: implications for vaccine design. Nat. Rev. Immunol. 4:630-40; Barouch, D. H. et al. (2002) Eventual AIDS vaccine failure in arhesus monkey by viral escape from cytotoxic lymphocytes. Nature 415:335-9.

Tuberculosis is caused by Mycobacterium tuberculosis. Smith, K. C. etal. (2008) Tuberculosis vaccines. In: Vaccines (Plotkin, S. A. et al.,eds.) 5th edition. 2008. Saunders Elsevier. The disease represents ahuge public health problem with approximately one third of the wordpopulation infected with the organism, despite widespread vaccinationprograms. Latent tuberculosis infection is the preclinical state of thedisease. Outbreak of disease can occur within weeks or decades from thetime of establishment of latent infection. Yearly deaths fromtuberculosis range in the millions. The exact immunological mechanismsthat underlie human resistance to M. tuberculosis remain to beelucidated. However, it is known that progressive disease is associatedwith a Th2 or a mixed Th1/Th2 response, whereas a pure Th1 responsecorrelates with protection. Surcel, H-M. et al. (1994) Th1/Th2 profilesin tuberculosis, based on the proliferation and cytokine response ofblood lymphocytes to mycobacterial antigens. Immunology 81: 171-6;Schauf, V. et al. (1993) Cytokine gene activation and modifiedresponsiveness to interleukin-2 in the blood of tuberculosis patients.J. Infect. Dis. 168: 1056-9. The Bacille Calmette-Guérin (BCG) vaccinesare the oldest vaccines currently in use. Unfortunately, the questionwhether the vaccines work has not been answered definitively. Efficaciesbetween 0 and 80% have been reported. The exact immune response elicitedby BCG vaccination as well as the mechanism of action within the hostare not well understood. Smith, K. C. et al. (2008). Animal studies havebeen infrequent. Smith, D. W. (1985) Protective effect of BCG inexperimental tuberculosis. Adv. Tuberc. Res. 22: 1-97. Nevertheless, theavailable information indicates that protective effects can betransferred with CD4 T-cells, but not with serum. Furthermore, theT-cell response is faster in vaccinated animals, resulting in more rapidmacrophage activation.

New and more effective vaccines are clearly needed. The presentinvention relates to vaccine compositions comprising areplication-competent controlled virus as well as to methods ofimmunization utilizing the latter compositions.

Replication-competent viruses and virus pairs controlled by a SafeSwitchor a SafeSwitch-like gene switch were disclosed generally in U.S. Pat.Nos. 7,906,312 and 8,137,947.

SUMMARY OF THE INVENTION

In one embodiment the present invention provides an improved vaccinecomprising a composition comprising an effective amount of areplication-competent controlled herpesvirus. The improved vaccineinduces superior protective immunity in a mammalian challenge model whencompared with a comparison vaccine comprising an equivalent amount of anattenuated herpesvirus that is replication-defective, provided that theattenuated herpesvirus and the replication-competent controlledherpesvirus have been derived from the same wild type virus. The latterwild type virus is also the challenge virus in the mammalian challengemodel, i.e., the protective immunity assessed is that directed to thiswild type virus. It is noted that improvement is established utilizing arelevant mammalian challenge model as human data are not yet available.A suitable mammalian challenge model is one in which infection ofanimals of the chosen mammalian species with the wild type virus ofinterest reproducibly results in a manifestation in the animals of arelevant aspect of the disease or condition induced in a human host byinfection with the same or a similar wild type virus. When available,small mammalian animal models are preferred, and mouse models are mostpreferred. “Protective immunity” is understood as an immune responsethat prevents or mitigates/reduces the severity or duration of thelatter manifestation of an aspect of the disease or condition that isinduced by inoculation with wild type virus, or reduces mortalityresulting therefrom.

The replication-competent controlled herpesvirus of the invention hasthe following set of properties:

(1) upon administration to a body region of a mammalian subject, thereplication-competent controlled virus remains essentiallynon-replicating in the body region in the absence of activation or,alternatively (i.e., in a different embodiment), replicates with asimilarly low efficiency as a replication-defective comparison virusadministered to a similar subject in a similar composition, in a similaramount and to a similar body region as the replication-competentcontrolled virus,(2) exposure of the body region to a localized activation treatmentactivates the replication-competent controlled virus to undergo a roundof replication in the body region, and(3) upon activation, the replication-competent controlled virusreplicates with an efficiency that is sufficient to induce in themammalian subject an immune response that is more effective in reducinginfection by the wild type virus from which the replication-competentcontrolled virus was derived and/or in reducing disease severity,disease duration or mortality subsequent to infection with said wildtype virus than the immune response induced in a similar subject by areplication-defective comparison virus (replication-competent controlledvirus and replication-defective comparison virus being administered insimilar compositions, in similar amounts and to similar body regions ofsimilar subjects).

The replication-defective comparison virus is a virus that has beenderived from the same wild type virus as the replication-competentcontrolled recombinant herpesvirus for which it is to serve as acomparison. Such a replication-defective comparison virus will bedisabled in at least one of the genes that are subjected to deliberateregulation in the replication-competent controlled virus. Disabledrefers to the result of a manipulation or event that renders the geneincapable of expressing a functional gene product. A wild type virus isa virus that has been isolated from a subject or the environment and,although propagated in vitro or in animals, has not been subjected toany deliberate selection or mutational process.

Efficiency of replication of a replication-competent controlledherpesvirus relative to a replication-defective comparison virus or thewild type virus from which the replication-competent controlled viruswas derived can be estimated by any method that allows fordeterminations of numbers of infectious virus (plaque-forming units orpfu) or numbers of viral particles. Biochemical methods for estimatingviral replication efficiency can also be utilized. These methods will beaimed at quantifying amounts of viral DNA, RNA or protein products, andmay include appropriate histochemical or immunohistochemical methods andbiochemical assays of amplification and detection of nucleic acids(e.g., realtime PCR, RT-PCR, probe hybridization) or detection of viralprotein products (e.g., western blot or other immunological assays). Anactivated replication-competent controlled virus would be said toreplicate with greater efficiency than a comparison virus if higherlevels of infectious virus or viral particles, or a biochemicalcorrelate such as viral DNA, were measured at a time point afterinoculation with equal numbers of infectious virus or viral particles atwhich (time point) the wild type virus from which the viruses beingcompared were derived would have completed essentially quantitativelyone replication cycle. A not-activated replication-competent controlledvirus would be said to replicate with lower efficiency than the wildtype virus from which the replication-competent controlled virus wasderived or a comparison virus if lower levels of infectious virus orviral particles, or a biochemical correlate such as viral DNA, weremeasured at a time point after inoculation with equal numbers ofinfectious virus or viral particles at which wild type virus would havecompleted essentially quantitatively at least one replication cycle. Ameasurement made at later time point would be accorded more weight thanone made at an earlier time (only in the case of a comparison involvinga not-activated replication-competent controlled virus). Typical invitro experiments for the determination of replication efficiencies ofactivated replication-competent controlled viruses are single stepgrowth curves. This type of experiment is also illustrated in theexample section.

The term “essentially non-replicating” used to characterize the in vivoreplication behavior of a not-activated replication-competent controlledvirus means that the not-activated replication-competent controlledvirus replicates with an efficiency that is at least 10 times lower thanthat of the wild type virus from which it was derived. More preferably,replication efficiency of the not-activated replication-competentcontrolled virus is at least 25 times lower than that of the wild typevirus from which it was derived. Even more preferably, replicationefficiency of the not-activated replication-competent controlled virusis at least 100 times lower than that of the wild type virus from whichit was derived.

The term “replicates with a similarly low efficiency as areplication-defective comparison virus” refers to a replicationefficiency of a not-activated replication-competent controlled virusthat is no more than 10 times that of the comparison virus, preferablyno more than 5 times that of the comparison virus, more preferably nomore than twice that of the comparison virus and, most preferably, notdetectably higher than that of the comparison virus. A five times higherreplication efficiency refers to a determination of a five times highernumber of not-activated replication-competent controlled virus or a fivetimes higher level of its biochemical correlate than that determined forthe comparison virus in the body region to which virus had beenadministered, whereby the determination is made at a time after the timepoint at which the wild type virus from which the viruses being comparedwere derived would have completed (essentially quantitatively) at leastone replication cycle.

A “similar subject” refers to a subject from the same species, same sexand about the same age. The term can be understood as having astatistical meaning, in which case a similar subject that receivescomparison virus is a group of subjects of the same mammalian species,of the same sex or mixture of sexes and of approximately same age or agedistribution as the group of subjects receiving replication-competentcontrolled virus.

“in a similar composition, in a similar amount and to a similar bodyregion” refers to a composition, amount or body region that is the sameas the composition, amount or body region it is compared to or is asclosely similar as can be achieved with a reasonable effort.

In specific embodiments, the vaccine compositions comprise areplication-competent controlled recombinant herpesvirus whose genomecomprises a gene for a small-molecule regulator-activated transactivatorwhich gene is functionally linked to a nucleic acid sequence that actsas a heat shock promoter or to a nucleic acid sequence that acts as aheat shock promoter as well as a transactivator-responsive promoter, andone or more transactivator-responsive promoters that are functionallylinked to one or more viral genes required for efficient replication,also referred to as a replication-essential genes. Subsequent toadministration of the virus to a body region of a mammalian subject, itcan be activated by subjecting the body region to an activating heatdose in the presence of an effective concentration in the body region ofan appropriate small-molecule regulator. Such an activation treatmentresults in one round of virus replication. For convenience, the latterreplication-competent controlled herpesviruses are referred to herein asheat- and small-molecule regulator-activated viruses. Thereplication-competent controlled herpesvirus can further comprise anexpressible heterologous gene (in addition to a gene encoding a(heterologous) transactivator) from another pathogen, an expressibleheterologous gene encoding an immune-modulatory polypeptide or anexpressible heterologous gene encoding another polypeptide, or anycombination of such genes. Such heterologous genes can be expressedunder the control of a transactivator-responsive promoter or of anotherpromoter, e.g., a constitutively active promoter. For example, aheterologous gene for an immune-modulatory polypeptide can be a geneencoding a cytokine or a chemokine. A heterologous gene can also be agene encoding a protein that facilitates local dissemination, infectionor replication of the virus such as, e.g., a gene for a matrixmetalloproteinase. A particular heterologous gene can be a gene for aprotein (antigen) of another pathogen (i.e., a pathogen other than thereplication-competent controlled virus or the wild type virus from whichit was derived). For example, a replication-competent controlledherpesvirus of the invention can contain inserted in its genome anexpressible gene for influenza surface antigen HA and/or NA. As afurther example, a heterologous gene may encode a modified genomic RNAof a retrovirus that lacks terminal repeat sequences and when expressedgives rise to viral particles that are replication-defective. It isconceivable that such viral particles will be more potent immunogensthan individually expressed viral proteins.

In other specific embodiments, the vaccine compositions comprise areplication-competent controlled recombinant herpesvirus whose genomecomprises a gene for a small-molecule regulator-activated transactivatorwhich gene is functionally linked to a nucleic acid sequence that actsas a constitutively active or a transactivator-enhanced promoter, anucleic acid sequence that acts as a heat shock promoter that isfunctionally linked to a first replication-essential viral gene, and atransactivator-responsive promoter that is functionally linked to asecond replication-essential viral gene. Subsequent to administration ofthe virus to a body region of a mammalian subject, it can be activatedby subjecting the body region to an activating heat dose in the presenceof an effective concentration in the body region of an appropriatesmall-molecule regulator. Such an activation treatment results in oneround of virus replication. The replication-competent controlledrecombinant virus can further comprise a heterologous gene from anotherpathogen, a heterologous gene encoding an immune-modulatory polypeptideor a heterologous gene encoding another polypeptide, or both. Suchheterologous genes can be expressed under the control of atransactivator-responsive promoter or of another promoter, e.g., aconstitutively active promoter. The latter replication-competentcontrolled herpesviruses are also referred to herein as heat- andsmall-molecule regulator-activated viruses. The term“transactivator-enhanced promoter” refers to a promoter that ofnecessity has some residual activity in the absence of transactivatorand whose activity increases as function of increasing levels oftransactivator (also termed an auto-activated promoter). A“transactivator-responsive promoter” is a promoter that ideally isinactive in the absence of the transactivator that activates it.

In yet other embodiments, the vaccine compositions comprise areplication-competent controlled recombinant herpesvirus whose genomecomprises a gene for a small-molecule regulator-activated transactivatorwhich gene is functionally linked to a nucleic acid sequence that actsas a constitutively active or a transactivator-enhanced promoter and atransactivator-responsive promoter that is functionally linked to areplication-essential viral gene. Subsequent to administration of thevirus to a body region of a mammalian subject, it can be activated bysubjecting the body region to an effective concentration of anappropriate small-molecule regulator. Such an activation treatmentresults in at least one round of virus replication. Thereplication-competent controlled virus can further comprise aheterologous gene from another pathogen, a heterologous gene encoding animmune-modulatory polypeptide or a heterologous gene encoding anotherpolypeptide, or both. Such heterologous genes can be expressed under thecontrol of a transactivator-responsive promoter or of another promoter,e.g., a constitutively active promoter. For convenience, the latterreplication-competent, controlled viruses are referred to herein assmall-molecule regulator-activated viruses.

In one embodiment, provided is a replication-competent controlledherpesvirus whose the genome comprises a gene for a small-moleculeregulator-activated transactivator which gene is functionally linked toa nucleic acid sequence that acts as a heat shock promoter or to anucleic acid sequence that acts as a heat shock promoter as well as atransactivator-responsive promoter, and one or moretransactivator-responsive promoters that are functionally linked to oneor more replication-essential viral genes wherein one of thereplication-essential viral genes is the ICP4 gene or the ICP8 gene ortwo of the replication-essential viral genes are the ICP4 and ICP8 genesif the replication-competent controlled herpesvirus is derived fromHSV-1 or HSV-2, or functional analogs or orthologs of these genes if thereplication-competent controlled herpesvirus is derived from anotherherpesvirus. Also provided is a vaccine composition comprising suchherpesvirus.

In one embodiment, provided is a replication-competent controlledherpesvirus whose the genome comprises a gene for a small-moleculeregulator-activated transactivator which gene is functionally linked toa nucleic acid sequence that acts as a constitutively active or atransactivator-enhanced promoter, a nucleic acid sequence that acts as aheat shock promoter that is functionally linked to a firstreplication-essential viral gene and a transactivator-responsivepromoter that is functionally linked to a second replication-essentialviral gene wherein the second replication-essential viral genes is theICP4 gene if the replication-competent controlled herpesvirus is derivedfrom HSV-1 or HSV-2, or a functional analog or ortholog of this gene ifthe replication-competent controlled herpesvirus is derived from anotherherpesvirus. Also provided is a vaccine composition comprising suchherpesvirus.

In one embodiment, provided is a composition comprising an effectiveamount of a replication-competent controlled herpesvirus for use ininducing protective immunity in a mammalian subject against the wildtype virus from which the replication-competent controlled herpesviruswas derived, whereby the genome of the replication-competent controlledherpesvirus comprises a gene for a small-molecule regulator-activatedtransactivator which gene is functionally linked to a nucleic acidsequence that acts as a heat shock promoter or to a nucleic acidsequence that acts as a heat shock promoter as well as atransactivator-responsive promoter and one or moretransactivator-responsive promoters that are functionally linked to oneor more replication-essential viral genes, and protective immunity isinduced by administering the composition to an area within orimmediately beneath the skin or mucosal membrane of the mammaliansubject, administering to the mammalian subject a composition comprisingan effective amount of small-molecule regulator and subjecting said areato a heat treatment that results in activation of thereplication-competent controlled herpesvirus.

A replication-competent controlled virus of the invention is derivedfrom a virus of the herpesviridae family. In more specific embodiments,the replication-competent controlled recombinant herpesvirus is derivedfrom a virus selected from an HSV-1, an HSV-2, a varicella zoster virus,a cytomegalovirus and a roseola virus.

Specific embodiments concern vaccine compositions comprising a heat- andsmall-molecule regulator-activated virus or a small-moleculeregulator-activated virus, whereby the virus is derived from an HSV-1 orHSV-2 and a transactivator-controlled replication-essential viral geneis all copies of the ICP4 gene or the ICP8 gene. In a more specificembodiment, the virus is identical with HSV-GS1 or is derived fromHSV-GS1. Further embodiments relate to compositions comprising a heat-and small-molecule regulator-activated virus or a small-moleculeregulator-activated virus, whereby the virus is derived from an HSV-1 orHSV-2 and a first transactivator-controlled replication-essential viralgene is all copies of the ICP4 gene and a secondtransactivator-controlled or heat shock promoter-drivenreplication-essential viral gene is the ICP8 gene. In a more specificembodiment, the virus is identical with HSV-GS3 or is derived fromHSV-GS3. In other embodiments, a heat- and small-moleculeregulator-activated virus or small-molecule regulator-activated virus isderived from an HSV-1 or HSV-2 and lacks a functional ICP47 gene. Thisvirus can be identical to or derived from HSV-GS4.

In particular embodiments of vaccine compositions comprising a heat- andsmall-molecule regulator-activated virus or a small-moleculeregulator-activated virus, the small-molecule regulator-activatedtransactivator contains a ligand-binding domain from a progesteronereceptor and is activated by a progesterone receptor antagonist or othermolecule capable of interacting with the ligand-binding domain and ofactivating the transactivator. Alternatively, it contains aligand-binding domain from an ecdysone receptor and is activated by anecdysteroid, a diacylhydrazine or other molecule capable of interactingwith the ligand-binding domain and of activating the transactivator. Inyet another alternative, it contains a ligand-binding domain from abacterial tetracycline repressor and is activated by a tetracycline orother molecule capable of interacting with the tetracycline repressordomain and of activating the transactivator. In yet another embodimentof compositions comprising a heat- and small-moleculeregulator-activated virus or a small-molecule regulator-activated virus,the small-molecule regulator-activated transactivator contains aligand-binding domain from an estrogen receptor and is activated by anestrogen receptor antagonist or other molecule capable of interactingwith the ligand-binding domain and of activating the transactivator. Ina further embodiment, the small-molecule regulator-activatedtransactivator is a complex of a polypeptide containing an FKBP12sequence and a polypeptide containing an FRB sequence from mTOR, and isactivated by rapamycin, a rapamycin derivative or other molecule capableof interacting with both polypeptides and of activating thetransactivator.

A vaccine composition of the invention comprising a heat- andsmall-molecule regulator-activated herpesvirus or a small-moleculeregulator-activated herpesvirus can further comprise a second virus thathas a host range which overlaps that of the replication-competentcontrolled virus and has a replication-essential gene functionallylinked to a transactivator-responsive promoter so that expression andactivation of the transactivator of the replication-competent controlledvirus also results in expression of the controlled replication-essentialgene of the co-infecting second virus, thereby enabling replication ofthe second virus. The immune response elicited by the latter compositionis expected to be directed not only against the replication-competentcontrolled virus but also against the second virus. The second virus canbe an adenovirus, and the replication-essential gene (of the secondvirus) that is functionally linked to a transactivator-responsivepromoter can be either the E1 or the E4 gene, or both of these genes.

Alternatively, a composition of the invention comprising a heat- andsmall-molecule regulator-activated herpesvirus or a small-moleculeregulator-activated herpesvirus can further comprise a second virus thathas a host range overlapping that of the replication-competentcontrolled virus, whereby the second virus lacks a replication-essentialgene or only contains a nonfunctional version thereof and, consequently,can only replicate, if the missing functional product of thereplication-essential gene is provided in trans. In this composition thereplication-competent controlled virus comprises a functional version ofthe latter replication-essential gene (of the second virus) which geneis functionally linked to a transactivator-responsive promoter so thatexpression and activation of the transactivator of thereplication-competent controlled virus also results in expression of thecontrolled replication-essential gene of the second virus, therebycomplementing the second virus. The immune response elicited by thelatter composition should not only be directed against thereplication-competent controlled virus but also against the secondvirus. The second virus can be an adenovirus having a nonfunctional E1and/or E4 gene and the replication-essential gene expressed from thereplication-competent controlled virus can be the E1 and/or E4 gene.

Other embodiments concern vaccine compositions comprising an effectiveamount of a heat- and small-molecule regulator-activated herpesvirus ora small-molecule regulator-activated herpesvirus and an effective amountof a small-molecule regulator that is capable of activating thetransactivator that controls replication of the heat- and small-moleculeregulator-activated herpesvirus or the small-moleculeregulator-activated herpesvirus.

The invention also relates to uses in immunization of a vaccinecomposition comprising an effective amount of a replication-competentcontrolled herpesvirus. In specific embodiments, the invention concernsthe use in immunization of a composition comprising an effective amountof a heat- and small-molecule regulator-activated herpesvirus or of asmall-molecule regulator-activated herpesvirus. The virus can compriseone or more of a heterologous gene from another pathogen, a heterologousgene encoding an immune-modulatory polypeptide or a heterologous geneencoding another polypeptide. Such heterologous genes can be expressedunder the control of a transactivator-responsive promoter or of anotherpromoter, e.g., a constitutively active promoter. Effective amounts ofvirus and small-molecule regulator can be present in the samecomposition. The latter composition is capable of inducing in amammalian subject to which it is administered and thereafter subjectedto activation treatment an immune response that is more effective inreducing infection by the wild type virus from which thereplication-competent controlled virus was derived and/or in reducingdisease severity, disease duration or mortality subsequent to infectionwith said wild type virus than the immune response induced in a similarsubject by a replication-defective comparison virus(replication-competent controlled virus and comparison virus beingadministered in similar compositions, in similar amounts and to similarbody regions of similar subjects).

The invention also encompasses methods of immunization in whichcompositions of the invention are administered to a subject. Oneembodiment of a method of immunization comprises (1) administering to abody region of a mammalian subject a composition of the inventioncomprising an effective amount of a heat- and small-moleculeregulator-activated herpesvirus which may also contain an expressibleheterologous gene, and (2) exposing said body region to an activatingheat dose in the presence in the body region of an effectiveconcentration of an appropriate small-molecule regulator (which is asmall-molecule regulator capable of activating the transactivator of theheat- and small-molecule regulator-activated herpesvirus). In a relatedmethod, effective amounts of virus and small-molecule regulator areco-administered in a single composition, and the body region is exposedto an activating heat dose.

In a related method, a composition of the invention comprising aneffective amount of a small-molecule regulator-activated herpesviruswhich may also contain an expressible heterologous gene is administeredto a body region, and the body region is exposed to an effectiveconcentration of small-molecule regulator. In a further related method,effective amounts of virus and small-molecule regulator areco-administered in a single composition. In derivative methods of theabove-described methods, a heat- and small-molecule regulator-activatedherpesvirus can be activated a second or further time by a second orfurther exposure of the body region (inoculation region) to anactivating heat dose in the presence of an effective concentration ofsmall-molecule regulator, or a small-molecule regulator-activated viruscan be re-activated by a second or further exposure of the body regionto an effective concentration of small-molecule regulator (provided thatthe second or further activation occurs prior to viral clearance).

The body region (inoculation site region) to which a composition of theinvention is administered can be any region on the surface of or withinthe body of a subject to which region an activating treatment, e.g., aheat dose and/or an effective dose of an appropriate small-moleculeregulator, can be administered. The body region can be a cutaneous orsubcutaneous region located anywhere on the trunk or the extremities ofthe subject. Preferably, administration of a composition can be to acutaneous or subcutaneous region located on an upper extremity of thesubject. Administration can also be to the lungs or airways, or a mucousmembrane in an orifice of a subject. Another preferred body region isthe nasal mucous membrane of a subject.

In one embodiment, provided is a method of immunization comprising: (1)administering to a body region of a mammalian subject a compositioncomprising an effective amount of a heat- and small-moleculeregulator-activated virus, and (2) exposing said body region to anactivating heat dose in the presence in the body region of an effectiveconcentration of an appropriate small-molecule regulator (which is asmall-molecule regulator capable of activating the transactivator of theheat- and small-molecule regulator-activated virus). In one embodiment,effective amounts of virus and small-molecule regulator areco-administered in a single composition, and the body region is exposedto an activating heat dose. In one embodiment, effective amounts ofvirus and small-molecule regulator are co-administered separately, andthe body region is exposed to an activating heat dose. In oneembodiment, the composition is any vaccine composition described herein.In one embodiment, the virus is any virus composition comprising areplication-competent controlled herpesvirus described herein. In oneembodiment, the method is a method of immunizing a subject against aherpetic disease or condition. In one embodiment, steps (1) and (2) arerepeated for one, two or more further cycles, optionally separated by atleast a week, a month, etc. Optionally, administration of thecomposition can be to a cutaneous or subcutaneous region located on anextremity of the subject.

In one embodiment, provided is a method of achieving substantially oneround of replication of a virus in a subject, comprising: (1)administering to a body region of a mammalian subject a compositioncomprising an effective amount of a heat- and small-moleculeregulator-activated herpesvirus, and (2) exposing said body region to anactivating heat dose in the presence in the body region of an effectiveconcentration of an appropriate small-molecule regulator (which is asmall-molecule regulator capable of activating the transactivator of theheat- and small-molecule regulator-activated herpesvirus). Optionally,multiple rounds of replication can be achieved by repeating step 2within one to several days after the preceding activating treatment. Inone embodiment, effective amounts of virus and small-molecule regulatorare co-administered in a single composition, and the body region isexposed to an activating heat dose. In one embodiment, effective amountsof virus and small-molecule regulator are co-administered separately,and the body region is exposed to an activating heat dose. In oneembodiment, the composition is any vaccine composition described herein.In one embodiment, the virus is any virus composition comprising areplication-competent controlled herpesvirus described herein. In oneembodiment, the method is a method of immunizing a subject against aherpetic disease or condition. Optionally, administration of thecomposition can be to a cutaneous or subcutaneous region located on anextremity of the subject.

In one embodiment, provided is a method of manufacturing areplication-competent controlled herpesvirus, the method comprising

(i) preparing a first precursor recombination vector by insertingnucleic acid sequences from a selected wild type herpesvirus flankingthe selected intragenic insertion site,

(ii) preparing a first recombination vector by inserting atransactivator gene cassette comprising a gene for a small-moleculeregulator-activated transactivator which gene is functionally linked toa nucleic acid sequence that acts as a heat shock promoter or to anucleic acid sequence that acts as a heat shock promoter as well as atransactivator-responsive promoter into the first precursorrecombination vector in a site between the flanking viral nucleotidesequences,(iii) introducing the gene cassette into the wild type herpesvirusgenome by recombination in a cell co-transduced with the firstrecombination vector and wild type virion DNA, identifying a firstrecombinant virus by detecting the presence of the gene cassette in thegenome of a progeny virus and preparing virion DNA therefrom,(iv) preparing a second precursor recombination vector by insertingnucleic acid sequences from the wild type herpesvirus flanking thepromoter region of a replication-essential viral gene selected forregulation,(v) preparing a second recombination vector by inserting atransactivator-responsive promoter into the second precursorrecombination vector in a site between the flanking viral nucleotidesequences,(vi) introducing the transactivator-responsive promoter into the genomeof the first recombinant virus by recombination in a cell co-transducedwith the second recombination vector and virion DNA of the firstrecombinant virus, identifying second recombinant virus by detecting thepresence of the transactivator-responsive promoter in the genome of aprogeny virus and preparing a stock of the progeny virus containing boththe transactivator gene cassette and a transactivator-regulatedreplication-essential viral gene. In one embodiment, thetransactivator-responsive promoter is inserted into the wild typeherpesvirus to produce a first recombinant virus and the transactivatorcassette is inserted into the first recombinant virus.

Any of the methods can further be characterized as comprising any stepdescribed in the application, including notably in the “DetailedDescription of the Invention”). The invention further relates to methodsof identifying, testing and/or making compositions described herein. Thedisclosure further relates to pharmaceutical, notably vaccine,formulations of the compositions disclosed herein. The disclosurefurther relates to methods of using the compositions in methods oftreatment or prevention of disease, e.g. a herpetic disease orcondition.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 presents an antiprogestin (mifepristone)-armed and heat-activated(or heat- and antiprogestin (mifepristone)-activated) SafeSwitchcontrolling a luciferase target gene. Reproduced from Vilaboa, N. andVoellmy, R. (2009) Deliberate regulation of therapeutic transgenes. In:Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, ThirdEdition (Smyth Templeton, N. ed.) CRC Press, Boca Raton, Fla., pp.619-36.

FIG. 2 relates to SafeSwitch performance in a stably transfected cellhuman line. Top panel: target gene activity one day after activatingheat treatment (HS) at 43° C. Bottom panel: gene activity 1 day (1 d)and 6 days (6 d) after heat activation (43° C./2 h) in the presence ofmifepristone (Mif), and reversibility of activation. *Mif was washedaway one day after HS. Reproduced from Vilaboa, N. and Voellmy, R.(2009).

FIG. 3 relates to single step growth experiments with HSV-GS1 in Verocells. (A) Controllability of replication. Four basic conditions weretested: (1) heat treatment at 43.5° C. for 30 min in the presence of 10nM mifepristone (activating treatment), (2) heat treatment alone, (3)mifepristone exposure alone, and (4) no treatment. Heat treatment wasadministered immediately after infection (i.e., immediately afterremoval of the viral inoculum). (B) Comparison of replicationefficiencies of wild type strain 17syn+ and HSV-GS1 with or withoutactivating treatment. Heat treatment was applied 4 h after infection.Mif: mifepristone. PFU/ml values and standard deviations are shown.

FIG. 4 relates to single step growth experiments with HSV-GS3. (A)Comparison of replication efficiencies of wild type strain 17syn+ andHSV-GS3 with or without activating treatment in E5 cells (17syn+) or E5cells transfected with an ICP8 expression plasmid (HSV-GS3). (B)Regulation of replication of HSV-GS3 in Vero cells. See the legend toFIG. 3 for a description of the four basic conditions tested (C)Analogous experiment in SCC-15 cells. In these experiments, heattreatments were administered immediately after infection. PFU/ml valuesand standard deviations are shown.

FIG. 5 relates to regulation of viral DNA replication and transcriptionin Vero cells infected with HSV-GS3. Multiple infected cultures weresubjected to the treatments indicated in the panels. Heat treatment(43.5° C. for 30 min) was administered 4 h after infection, and sets ofcultures were harvested 1, 4, 12 and 24 h later, and DNA and RNA wereextracted and analyzed by qPCR and RT-qPCR, respectively. Uli:ulipristal. (A) HSV DNA. (B) ICP4 RNA. (C) gC RNA. Values and standarddeviations were normalized relative to the highest value in each panel.

FIG. 6 relates to regulation of HSV-GS3 DNA replication andtranscription, and comparison of replicative yields between HSV-GS3 andKD6 in the mouse footpad model. Adult outbred mice were inoculated onthe slightly abraded footpads of their hind legs with 1×10⁵ PFU ofHSV-GS3 or KD6. Indicated doses of ulipristal were administeredintraperitoneally at the time of infection. Localized heat treatment at45° C. for 10 min was performed 3 h after virus administration. Micewere sacrificed 24 h (panels A-C) or 4 days (panel D) post heattreatment, and DNA and RNA were isolated from feet and dorsal rootganglia (DRG) and analyzed by qPCR and RT-qPCR, respectively. (A) HSVDNA. (B) ICP4 RNA. (C) gC RNA. (D) HSV DNA at 4 days post heattreatment. Values and standard deviations were normalized relative tothe highest value in each panel. ND: none detected.

DETAILED DESCRIPTION

Unless otherwise defined below or elsewhere in the presentspecification, all terms shall have their ordinary meaning in therelevant art.

“Replication of virus” or “virus/viral replication” are understood tomean multiplication of viral particles. Replication is often measured bydetermination of numbers of infectious virus, e.g., plaque-forming unitsof virus (pfu). However, replication can also be assessed by biochemicalmethods such as methods that determine amounts of viral DNA, e.g., by arealtime PCR procedure, levels of viral gene expression, e.g., by RT-PCRof gene transcripts, etc. However, it is understood that marginalincreases in levels of viral DNA or viral gene transcripts or proteinproducts may not translate in corresponding marginal increases in virusreplication due to threshold effects.

“Proteotoxic stress” is a physical or chemical insult that results inincreased protein unfolding, reduces maturation of newly synthesizedpolypeptides or causes synthesis of proteins that are unable to foldproperly.

A “small-molecule regulator” is understood to be a low molecular weightligand of a transactivator used in connection with this invention. Thesmall-molecule regulator is capable of activating the transactivator.The small-molecule regulator is typically, but not necessarily, smallerthan about 1000 Dalton (1 kDa).

The term “transactivator” is used herein to refer to a non-viral and,typically, engineered transcription factor that when activated by asmall-molecule regulator can positively affect transcription of a genecontrolled by a transactivator-responsive promoter.

“Activated” when used in connection with a transactivated gene meansthat the rate of expression of the gene is measurably greater afteractivation than before activation. When used in connection with atransactivator, “active” or “activated” refers to atransactivation-competent form of the transactivator.

“Promoter of a heat shock gene”, “heat shock gene promoter” and “heatshock promoter” are used synonymously. A “nucleic acid that acts as aheat shock promoter” can be a heat shock promoter or a nucleic acid thatcontains sequence elements of the type present in heat shock promoterswhich elements confer heat activation on a functionally linked gene.

Herein, a virus, whose genome includes a foreign (heterologous)non-viral or viral gene, is either referred to as a “virus” or a “viralvector”.

A “replication-competent controlled virus” is a recombinant virus whosereplicative ability is under the control of a gene switch that can bedeliberately activated.

A “recombinant virus” refers specifically to a virus that has beenaltered by an experimenter. Often, a “recombinant virus” is simplyreferred to as a “virus”.

A “replication-essential gene” or a “gene required for efficientreplication” is arbitrarily defined herein as a viral gene whose loss offunction diminishes replication efficiency by a factor of ten orgreater. Replication efficiency can be estimated, e.g., in a single stepgrowth experiment. For many viruses it is well known which genes arereplication-essential genes. For herpesviruses see, e.g., Nishiyama, Y.(1996) Herpesvirus genes: molecular basis of viral replication andpathogenicitiy. Nagoya L. Med. Sci. 59: 107-19.

An “effective amount of a replication-competent controlled virus” is anamount of such virus that upon single or repeated administration to asubject followed by activation detectably enhances the subject'sresistance to infection by the wild type virus from which thereplication-competent controlled virus was derived and/or detectablyreduces disease severity, disease duration or mortality subsequent toinfection with said wild type virus. A corresponding amount of areplication-defective comparison virus, administered in a similarcomposition and to a similar body region of a similar subject induces alower level of such functional immunity.

An “effective amount of a small-molecule regulator” is an amount thatwhen administered to a subject by a desired route is capable ofco-activating (in combination with a heat treatment) a heat- andsmall-molecule regulator-activated virus or activating a small-moleculeregulator-activated virus with which the subject concurrently is, hasbeen or will be inoculated to undergo a round of replication (or atleast one round of replication in the case of a small-moleculeregulator-activated virus) in the inoculation site region.

A “subject” or a “mammalian subject” is a mammalian animal or a humanperson.

A “heat shock gene” is defined herein as any gene, from any eukaryoticorganism, whose activity is enhanced when the cell containing the geneis exposed to a temperature above its normal growth temperature.Typically, such genes are activated when the temperature to which thecell is normally exposed is raised by 3-10° C. Heat shock genes comprisegenes for the “classical” heat shock proteins, i.e., Hsp110, Hsp90,Hsp70, Hsp60, Hsp40, and Hsp20-30. They also include otherheat-inducible genes such as genes for MDR1, ubiquitin, FKBP52, hemeoxidase and other proteins. The promoters of these genes, the “heatshock promoters”, contain characteristic sequence elements referred toas heat shock elements (HSE) that consist of perfect or imperfectsequence modules of the type NGAAN or AGAAN, which modules are arrangedin alternating orientations (Amin, J. et al. (1988) Key features of heatshock regulatory elements. Mol. Cell. Biol 8: 3761-3769; Xiao, H. andLis, J. T. (1988) Germline transformation used to define key features ofheat-shock response elements. Science 239: 1139-1142; Fernandes, M. etal. (1994) Fine structure analyses of the Drosophila and Saccharomycesheat shock factor—heat shock element interactions. Nucleic Acids Res.22: 167-173). These elements are highly conserved in all eukaryoticcells such that, e.g., a heat shock promoter from a fruit fly isfunctional and heat-regulated in a frog cell (Voellmy, R. and Rungger,D. (1982) Transcription of a Drosophila heat shock gene is heat-inducedin Xenopus oocytes. Proc. Natl. Acad. Sci. USA 79: 1776-1780). HSEsequences are binding sites for heat shock transcription factors (HSFs;reviewed in Wu, C. (1995) Heat shock transcription factors: structureand regulation. Annu. Rev. Cell Dev. Biol. 11, 441-469). The factorprimarily responsible for activation of heat shock genes in vertebratecells exposed to heat or a proteotoxic stress is heat shocktranscription factor 1 (referred to herein as “HSF1”) (Baler, R. et al.(1993) Activation of human heat shock genes is accompanied byoligomerization, modification, and rapid translocation of heat shockfactor HSF1. Mol. Cell. Biol. 13: 2486-2496; McMillan, D. R. et al.(1998) Targeted disruption of heat shock factor 1 abolishesthermotolerance and protection against heat-inducible apoptosis. J.Biol. Chem. 273, 7523-7528). Preferred promoters for use inreplication-competent controlled viruses discussed herein are those frominducible hsp70 genes. A particularly preferred heat shock promoter isthe promoter of the human hsp70B gene (Voellmy, R. et al. (1985)Isolation and functional analysis of a human 70,000-dalton heat shockprotein gene fragment. Proc. Natl. Acad. Sci. USA 82, 4949-4953).

“Vaccine” typically refers to compositions comprising microorganismsthat are killed, replication-defective or otherwise attenuated. Herein,the term is expanded to also include compositions comprisingreplication-competent controlled viruses that can induce an immuneresponse in the subject to which they are administered.

As was alluded to under Background, current thought appears to be thatin order to be effective or more effective, respectfully, improvedvaccine candidates for preventing or treating diseases such as herpes,HIV, tuberculosis or influenza need to elicit a balanced immune responsethat also includes a powerful effector T cell response. The presentinvention relates to replication-competent controlled viruses that, uponactivation, replicate with efficiencies that approach those of therespective wild type viruses. The inventors hypothesized that theserecombinant viruses and any heterologous protein they express will bepotent immunogens that elicit balanced immune responses.

The proposed novel approach for immunization intends to bring to bear,in a carefully controlled fashion, the unattenuated replicativepotential of the chosen viral vector. The underlying concept may beillustrated by reference to a historical approach for smallpoxprevention known as variolation. Flower, D. R. (2008) Bioinformatics forvaccinology. John Wley & Sons; Henderson, D. A. et al. (2008) Smallpoxand vaccinia. In: Vaccines (Plotkin, S. A. et al., eds.) 5th edition.2008. Saunders Elsevier. The procedure that predates the first vaccinerelates to the inoculation of a healthy person, typicallysubcutaneously, with dried scabs or pustular fluid from a personrecovering from smallpox. When it was practiced routinely (until thebeginning of the 19^(th) century in Europe and the United States, anduntil at least the second half of the 20^(th) century in remote parts ofAfrica and Asia), the procedure had a post-infection fatality rate ofabout 0.5-2%, which compared favorably with a mortality rate caused bythe disease of about 20-30%. Variolation was effective in rendering aperson immune against smallpox. While there may be several reasons whythis procedure worked as well as it did, one appears to be related tothe choice of inoculation site: smallpox (i.e., Variola virus) normallyenters the lungs in the form of small aerosol droplets. From there thevirus spreads rapidly. When inoculated subcutaneously, progress is lessrapid, allowing the immune system to catch up with the disease at anearly stage. Because of the substantial post-infection fatality rate,the variolation procedure was rapidly superseded by Edward Jenner'svaccination method when it became generally available. It is furthernoted that variolation suffered from another serious problem. Variolatedpersons were infectious for a certain period of time. During thisperiod, especially since they typically were not or only mildly ill,they were capable of moving about and spreading the virus to naïvepersons.

The general procedure exemplified by variolation of inducing immunity ina subject by administering a disease-causing microbial agent to aninnocuous local site and allowing the agent to vigorously proliferatelocally for a defined period of time so that strong innate and adaptiveimmune responses are triggered, could be made safe if (1) it could beassured that proliferation of the immunizing viral agent is restrictedeffectively to the chosen inoculation region as well as is narrowlylimited in time, and (2) the possibility of recrudescence of significantvirus replication at the inoculation site or elsewhere could beprevented and (3) the possibility of dissemination of disease-causingvirus by immunized subjects could be excluded. The novel immunizationmethod involving replication-competent controlled viruses discussedherein has been developed with the specific aim of providing thisrequired safety. A wild type virus is genetically altered by placing atleast one selected replication-essential gene under the control of agene switch that has a broad dynamic range, i.e., that essentiallyfunctions as an on/off switch. Most preferred are heat- andsmall-molecule regulator-activated (dual-responsive) gene switches thatwere discussed, e.g., in Vilaboa, N. et al. (2011) Gene switches fordeliberate regulation of transgene expression: recent advances in systemdevelopment and uses. J. Genet. Syndr. Gene Ther. 2: 107. A particulargene switch of this kind, referred to as SafeSwitch (co-activated byheat and an antiprogestin), has been used in Examples and is illustratedin FIG. 1. Unless specifically indicated, the description that followsrelates to viruses whose replication has been brought under the controlof such a dual-responsive gene switch. However, the description is alsorelevant to other replication-competent controlled viruses of theinvention (i.e., other heat- and small-molecule regulator-activatedviruses and small-molecule regulator-activated viruses).

Replication of the so modified virus, a replication-competent controlled(recombinant) virus, only occurs when the dual-responsive gene switch isarmed by an appropriate small-molecule regulator as well as is triggeredby transient heat treatment (at a level below that causing burns or painbut above that which may be encountered in a feverish patient). Once thegene switch is activated, the virus expresses the full complement ofviral proteins (or the desired complement of viral proteins and,optionally, heterologous RNA or proteins) and replicates with anefficiency approaching that of wild type virus.

Heat can be readily focused. Administering focused heat to a body regionto which replication-competent controlled virus has been administered(i.e., the inoculation site) to trigger activation of thedual-responsive gene switch (in the presence of small-moleculeregulator) will result in virus replication that is strictly confined tothe heated region. The dual requirement for heat and a small-moleculeregulator is intended to provide a high level of security againstaccidental virus replication. In the absence of small-moleculeregulator, activation/re-activation of virus is virtually impossible.Similarly, in the absence of a concomitant heat treatment, virusreplication is not normally activated.

The arming small-molecule regulator needs to satisfy a number ofcriteria. Most important will be that the substance is safe; adverseeffects should occur at most at an extremely low rate and should begenerally of a mild nature. Ideally, the chosen small-molecule regulatorwill belong to a chemical group that is not used in human therapy.However, before any substance not otherwise developed for human therapycould be used as small-molecule regulator in an immunization procedure,it would have to undergo extensive preclinical and clinical testing. Itmay be more efficacious to select a known and well-characterized drugsubstance that is not otherwise administered to the specific populationtargeted for immunization. Alternatively, a known drug substance may beselected that (1) will not need to be administered to subjects within atleast the first several weeks after immunization and (2) is indicatedonly for short-term, sporadic administration, preferably under medicalsupervision. Thus, a potential low-level risk is further reduced by theavoidance of administration of the drug substance during the periodduring which immunizing virus is systemically present. Sporadic use ofthe drug substance under medical supervision will ensure that anysignificant inadvertent replication of immunizing virus would be rapidlydiagnosed and antiviral measures could be taken without delay. In theexample systems described herein, the arming small-molecule regulator isa progesterone receptor (PR) antagonist or antiprogestin, e.g.,mifepristone or ulipristal. Mifepristone and ulipristal fulfill thelatter requirements of not typically needing to be administered shortlyafter immunization, and of being used only infrequently (and only in aspecific segment of the population) and only under medical supervision.Mifepristone and ulipristal have excellent safety records.

How the novel immunization method can be practiced is illustrated in thefollowing specific example. A composition comprising an effective amountof a replication-competent controlled virus and an effective amount of asmall-molecule regulator is administered to a subject intradermally orsubcutaneously. Shortly after administration, a heating patch isactivated and applied to the inoculation site by either the subject orthe physician. Heating at about 43.5-45.5° C. (temperature of the patchsurface in contact with the skin) will be for a period of about 10-60min. The latter heat treatment will trigger one cycle of virusreplication. If another round of replication is desired, anotheractivated patch is applied to the inoculation site at an appropriatelater time. If an immunization procedure involves sequential heattreatments, small-molecule regulator may also need to be administeredsequentially. Alternatively, a slow release formulation may be utilizedthat assures the presence of an effective concentration ofsmall-molecule regulator in the inoculation site region over the periodduring which viral replication is desired.

More generally, a body region to which a replication-competentcontrolled virus of the invention is administered, i.e., the inoculationsite region, may be heated by any suitable method. Heat may be deliveredor produced in the target region by different means including directcontact with a heated surface or a heated liquid, ultrasound, infraredradiation, or microwave or radiofrequency radiation. As proposed in theabove specific example, a practical and inexpensive solution may beoffered by heating patches (or similar devices of other shapes, e.g.,cylinders or cones, for heating mucosal surfaces of the nose, etc.)containing a supercooled liquid that can be triggered by mechanicaldisturbance to crystallize, releasing heat at the melting temperature ofthe chemical used. A useful chemical may be sodium thiosulfatepentahydrate that has a melting temperature of about 48° C. U.S. Pat.Nos. 3,951,127, 4,379,448, and 4,460,546. The technology is readilyavailable and is already being used in a number of health care products.

An “activating heat dose” is a heat dose that causes a transientactivation of HSF1 in cells within the inoculation site region.Activation of this transcription factor is evidenced by a detectablyincreased level of RNA transcripts of a heat-inducible heat shock geneover the level present in cells not exposed to the heat dose.Alternatively, it may be evidenced as a detectably increased amount ofthe protein product of such a heat shock gene. Moreover, an activatingheat dose may be evidenced by the occurrence of replication ofreplication-competent controlled virus in the presence of an effectiveconcentration of an appropriate small-molecule regulator.

An activating heat dose can be delivered to the target region at atemperature between about 41° C. and about 47° C. for a period ofbetween about 1 min and about 180 min. It is noted that heat dose is afunction of both temperature and time of exposure. Hence, similar heatdoses can be achieved by a combination of an exposure temperature at thelower end of the temperature range and an exposure time at the upper endof the time range, or an exposure temperature at the higher end of thetemperature range and an exposure time at the lower end of the timerange. Preferably, heat exposure will be at a temperature between about42° C. and about 46° C. for a period of between about 5 min and about150 min. Most preferably, heat treatment is administered at atemperature between about 43.5° C. and about 45.5° C. for a period ofbetween about 10 min and about 60 min.

An effective concentration of a small-molecule regulator in theinoculation site region is a concentration that enables replication (oneround) of replication-competent controlled virus in infected cells ofthat region that have also received an appropriate heat dose. What aneffective concentration is depends on the affinity of the small-moleculeregulator for its target transactivator. How such effectiveconcentration is achieved and for how long it is maintained also dependson the pharmacokinetics of the particular small-molecule regulator,which in turn depends on the route of administration of thesmall-molecule regulator, the metabolism and route of elimination of thesmall-molecule regulator, the subject being examined, i.e., the type ofsubject (human or other mammal), its age, condition, weight, etc. Itfurther depends on the type of composition administered, i.e., whetherthe composition permits an immediate release or a slow release of theregulator. For a number of well-characterized small-moleculeregulator-transactivator systems, effective concentrations in certainexperimental subjects have been estimated and are available from theliterature. This applies to systems based on progesterone receptor,ecdysone receptors, estrogen receptors, and tetracycline repressor aswell as to dimerizer systems, i.e., transactivators activated byrapamycin or analogs (including non-immunosuppressive analogs), or FK506or analogs. For example, an effective concentration of mifepristone inrats can be reached by i.p. (intraperitoneal) administration of aslittle as 5 μg mifepristone per kg body weight (5 μg/kg). Amounts wouldhave to be approximately doubled (to about 10 μg/kg), if thesmall-molecule regulator is administered orally. Wang, Y. et al. (1994)A regulatory system for use in gene transfer. Proc. Natl. Acad. Sci. USA91: 8180-84. Such amounts of a small-molecule regulator that, uponadministration by the chosen route, result in an effective concentrationare referred to as effective amounts of the small-molecule regulator inquestion. How an effective amount of a small-molecule regulator thatresults in an effective concentration can be determined is well withinthe skills of an artisan and is also addressed in the example section.

In the afore-described specific example of how the novel immunizationmethod may be practiced, a replication-competent controlled virus of theinvention (a heat- and small-molecule regulator-activated virus) and anappropriate small-molecule regulator were co-administered in a singlecomposition. Replication-competent controlled virus and small-moleculeregulator can also be administered in separate compositions. Topicalco-administration of immunizing virus and small-molecule regulatorappears advantageous for several reasons, including minimization ofpotential secondary effects of the small-molecule regulator, furtherreduction of the already remote possibility that virus may replicatesystemically during the immunization period, and minimization of theenvironmental impact of elimination of small-molecule regulator.Notwithstanding these advantages, the small-molecule regulator may begiven by a systemic route, e.g., orally, which may be preferred if aformulation of the drug substance of choice is already available thathas been tested for a particular route of administration. The relativetiming of inoculation with replication-competent controlled virus,administration of an appropriate heat dose and administration of aneffective amount of small-molecule regulator is derivative of theoperational requirements of dual-responsive gene switch control.Typically, inoculation with immunizing virus will precede heattreatment. This is because heat activation of heat shock transcriptionfactor (HSF1) is transient, and activated factor returns to an inactivestate within at most a few hours after activation. The dual-responsivetransactivator gene present in the viral genome must be available forHSF1-mediated transcription during the latter short interval of factoractivity. For the latter gene(s) to become available for transcription,the immunizing virus will have had to adsorb to a host cell, enter thecell and unravel to present its genome to the cellular transcriptionmachinery. Although not preferred, it is possible to heat-expose theinoculation site region immediately after (or even shortly before)administration of the immunizing virus. Typically, the inoculation siteregion is heat-exposed at a time between about 30 min to about 10 hafter virus administration, although heat treatment may be administeredeven later. Regarding administration of the small-molecule regulator,there typically will be more flexibility because it will be possible tomaintain an effective concentration systemically or specifically in theinoculation site region for one to several days. Consequently,small-molecule regulator can be administered prior to, at the time of orsubsequent to virus administration, the only requirement being that theregulator be present in an effective concentration in the inoculationsite region for the time needed for the target transactivator to fulfillits role in enabling viral replication. Typically, this time willcorrespond to that required for the completion of a round of inducedvirus replication. Typically, a round of virus replication will becompleted within about one day.

As has been alluded to before, in the novel immunization method areplication-competent controlled virus may be induced to replicate onceor several times. Replication may be re-induced one to several daysafter the previous round of replication. Such repeated replication willserve to increase viral load in the subject. For any round ofreplication to occur, the target cells that are infected withreplication-competent controlled virus need to receive an activatingheat dose and the tissue of which the latter cells are part (theinoculation region) must contain an effective concentration ofsmall-molecule regulator.

Immunization can be by any suitable route, provided that a fraction ofadministered replication-competent controlled virus infects cells withina narrowly defined region that can be subjected to focused/localizedheat treatment, and replication of the virus in the latter cellstriggers the desired immune response (without causing disease or unduediscomfort). The body region (inoculation site region) to whichimmunizing virus is administered may be a cutaneous or subcutaneousregion located anywhere on the trunk or the extremities of the subject.Preferably, administration of a composition of the invention comprisinga replication-competent controlled virus may be to a cutaneous orsubcutaneous region located on an upper extremity of the subject.Administration may also be to the lungs or airways, or a mucous membranein an orifice of a subject. This includes the nasal mucous membrane of asubject.

Dual-responsive gene switches consist of (1) a gene for a small-moleculeregulator-activated transactivator, the gene being functionally linkedto a promoter or promoter cassette responsive to heat and thetransactivator and (2) a promoter responsive to the transactivator forcontrolling a gene of interest. Vilaboa, N. et al. (2005) Novel geneswitches for targeted and timed expression of proteins of interest. Mol.Ther. 12: 290-8. FIG. 1 shows an example dual-responsive gene switch(the specific gene switch being referred to as “SafeSwitch”)incorporating mifepristone (and ulipristal)-dependent chimerictransactivator GLp65 (or glp65). This transactivator comprises aDNA-binding domain from yeast transcription factor GAL4, a truncatedligand-binding domain from a human progesterone receptor and atransactivation domain from the human RelA protein. Burcin, M. M. et al.(1999) Adenovirus-mediated regulable target gene expression in vivo.Proc. Natl. Acad. Sci. USA 96: 355-60; Ye, X. et al. (2002)Ligand-inducible transgene regulation for gene therapy. Meth. Enzymol.346: 551-61. In a cell containing gene switch and target gene, thetarget gene is not expected to be expressed in the absence ofsmall-molecule regulator, e.g., mifepristone, or an activating heattreatment. When the cell is subjected to a heat treatment of anappropriate intensity, (endogenous) heat shock transcription factor 1(HSF1) is activated, and transcription from the transactivator gene isinitiated. A short time later, HSF1 is de-activated, and HSF1-drivenexpression of transactivator ceases. In the absence of small-moleculeregulator, transactivator synthesized remains inactive and is eventuallyremoved by degradation. However, in the presence of small-moleculeregulator, transactivator is activated and mediates expression from itsown gene as well as from the target gene. As a consequence, a certaintransactivator level is maintained and target protein continues to besynthesized (theoretically) as long as small-molecule regulator remainspresent. Its withdrawal/removal causes inactivation of transactivator.Target as well as transactivator gene expression will diminish andeventually cease, and the system will reset itself.

The mifepristone-armed and heat-activated SafeSwitch was testedextensively in vitro in transient transfection and stable cell lineformats and in vivo after electroporation of gene switch and target geneinto mouse gastrocnemius muscle. Vilaboa et al. (2005). Results obtainedfrom experiments with a cell line stably containing the gene switch anda luciferase target gene are reproduced in FIG. 2. The data reveal thatthe system performed as intended. Essentially no target proteinexpression occurred in the absence of mifepristone, even when cells weresubjected to an activating heat treatment. Heat treatment in thepresence but not in the absence of mifepristone resulted in activationof target gene expression. It is noted that the heat threshold wasrelatively elevated: even a 1-h heat treatment at 43° C. only resultedin submaximal activation. Activation of target gene expression wasclearly heat dose-dependent. A single heat treatment induced sustainedtarget gene expression for at least 6 days, but only in the continuedpresence of mifepristone. Removal of mifepristone subsequent toactivation resulted in cessation of target gene expression (as evidencedby a disappearance of the labile target gene product).

Analogous dual-responsive gene switches that are activated by heattreatment in the presence of rapamycin or a non-immunosuppressiverapamycin derivative were also developed. Martin-Saavedra, F. M. et al.(2009) Heat-activated, rapamycin-dependent gene switches for tightcontrol of transgene expression. Hum. Gene Ther. 20: 1060-1. Twodifferent versions were prepared that are capable of transactivating atarget gene driven by a promoter containing ZFHD1-binding sites (asoriginally described in Rivera, V. M. et al. (1996) A humanized systemfor pharmacologic control of gene expression. Nat. Med. 2: 1028-32) or aGAL4 promoter, respectively.

Other examples for small-molecule regulator-activated transactivatorsthan can be incorporated in dual-responsive gene switches or relatedgene switches include tetracycline/doxycycline-regulated tet-onrepressors (Gossen, M. and Bujard, H. (1992) Tight control of geneexpression in mammalian cells by tetracycline-responsive promoters.Proc. Natl. Acad. Sci. USA 89, 5547-5551; Gossen, M. et al. (1996)Transcriptional activation by tetracyclines in mammalian cells. Science268, 1766-1769) and transactivators containing a ligand-binding domainof an insect ecdysone receptor (No, D. et al. (1996) Ecdysone-induciblegene expression in mammalian cells and transgenic mice. Proc. Natl.Acad. Sci. USA 93, 3346-3351). A stringently ligand-dependenttransactivator of the latter type is the RheoSwitch transactivatordeveloped by Palli and colleagues. Palli, S. R. et al. (2003) Improvedecdysone receptor-based inducible gene regulation system. Eur. J.Biochem. 270: 1308-15; Kumar, M. B. et al. (2004) Highly flexible ligandbinding pocket of ecdysone receptor. A single amino acid change leads todiscrimination between two groups of nonsteroidal ecdysone agonists. J.Biol. Chem. 279: 27211-18. The RheoSwitch transactivator can beactivated by ecdysteroids such as ponasterone A or muristerone A, or bysynthetic diacylhydrazines such as RSL-1 (also known as RH-5849, firstsynthesized by Rohm and Haas Company). Dhadialla, T. S. et al. (1998)New insecticides with ecdysteroidal and juvenile hormone activity. Annu.Rev. Entomol. 43: 545-69. Other small molecule-regulated transactivatorsmay be used, provided that they can be employed to control the activityof a target gene without also causing widespread deregulation of genesof the host cell and provided further that the associated small-moleculeregulators have acceptably low toxicity for the host at their effectiveconcentrations.

Gene switches related to the above-discussed dual-responsive geneswitches consist of (1) a gene for a small-molecule regulator-activatedtransactivator, the gene being functionally linked to a heat-responsivepromoter, and (2) a promoter responsive to the transactivator forcontrolling a gene of interest. Unlike in the above-discusseddual-responsive gene switches, the transactivator gene is notauto-activated. As a consequence, the period of activity of such a geneswitch subsequent to a single activating heat treatment is substantiallyshorter than that of a corresponding dual-responsive gene switchcontaining an auto-activated transactivator gene.

The novel immunization approach is exemplified herein by HSV-1-derivedviruses. Other viruses including other types of herpesviruses can beemployed as backbone for a replication-competent controlled virus of theinvention. These include the alpha herpesviruses HSV2 and varicellazoster virus (VZV), beta herpesviruses including cytomegalovirus (CMV)and the roseola viruses (HSV6 and HSV7), and gamma herpesviruses such asEpstein-Barr virus (EBV) and Karposi's sarcoma-associated herpesvirus(KSHV). Preferred replication-competent controlled viruses of theinvention are derived from HSV-1, HSV2 or varicella zoster viruses(VZV).

In a different embodiment, two replication-essential genes of areplication-competent controlled virus are not controlled by adual-responsive gene switch that places them under dual control of heatand a small-molecule regulator, but are individually controlled by aheat shock promoter and a transactivator-responsive promoter (at leastone replication-essential gene being controlled by a heat shock promoterand at least one replication-essential gene being controlled by atransactivator-responsive promoter). Transactivator is expressed from aconstitutive promoter or from an auto-activated(transactivator-enhanced) promoter. If one replication-essential gene iscontrolled by a heat shock promoter and another by a promoter responsiveto an activated transactivator, replication is also dually controlled byheat and a small-molecule regulator. However, a replication-competentcontrolled virus of this type is less preferred for several reasons.First, activated HSF1 and, consequently, heat shock promoters tend to beinactivated within a period of a few hours. Hence, if expressed underheat shock promoter control, certain viral genes may not be capable offulfilling their normal role in the virus replication cycle. Second,also related to the transient nature of the heat shock response, ifexpression of two differently regulated viral genes (i.e., regulated byheat shock or transactivator, respectively) is required at differenttimes in the virus life cycle, activating heat treatment andsmall-molecule regulator may need to be administered at different times,adding considerable inconvenience to an immunization procedure. Arequirement that only genes that exhibit closely similar expressionprofiles be selected for regulation would represent a significantconstraint on the design of a controlled virus. Finally, in the presenceof one of the activating stimuli, i.e., heat or small-moleculeregulator, one of the two differently regulated replication-essentialgenes will become activated, weakening the replication block. In thecase where both replication-essential genes are dually regulated,neither of the genes will become active in the presence of ligand or ifthe host cell is exposed to heat. Hence, the stringent inhibition ofreplication will be maintained even in the presence of one of theactivating stimuli. Regarding an appropriate heat dose for activation,nature and properties of transactivators, and properties and effectiveconcentrations of virus and small-molecule regulators, etc., the readeris referred back to earlier sections of this specification.

In yet another less preferred embodiment, one or morereplication-essential genes of a replication-competent controlled virusare not dually controlled by heat and a small-molecule regulator, butare singly controlled by a small-molecule regulator. A gene for asmall-molecule regulator-activated transactivator is expressed from aconstitutive promoter or from an auto-activated promoter. To achieve adegree of localization of virus replication, small-molecule regulator isalso administrated to the inoculation site, either together with theimmunizing virus or separately. A slow release formulation may beutilized that assures the presence of an effective concentration ofsmall-molecule regulator in the inoculation site region for the periodduring which viral replication is desired. Regarding the nature andproperties of transactivators, and properties and effectiveconcentrations of virus and small-molecule regulators, etc., the readeris referred back to earlier sections of this specification.

The replication-competent controlled viruses of the invention can alsobe utilized as vectors to deliver antigens from another infectiousagent. Viruses of the herpesviridae family can accommodate sizeable DNAinsertions in their genome, which insertions are not expected to reducesignificantly replication efficiency. Inserted genes encoding, e.g.,influenza virus surface antigens or internal proteins, HIV envelope orinternal antigens, etc., may be subjected to heat and/or small moleculeregulator control. This would link antigen expression to virusreplication and restrict it to the inoculation site region.Alternatively, inserted genes may be placed under the control of otherpromoters, e.g., constitutive promoters, allowing for expression innon-productively infected cells, which would result in longer periods ofantigen expression as well as expression in virus-infected cells outsideof the inoculation site region.

Viruses have long been used as vehicles for delivery of antigens ofanother pathogen. Smith, G. L. et al. (1983) Infectious vaccinia virusrecombinants that express hepatitis B virus surface antigen. Nature 302:490-5; Moss, B. et al. (1984) Live recombinant vaccinia virus protectschimpanzees against hepatitis B. Nature 311: 67-9. Attenuated viralvectors employed included replication-defective viruses as well asviruses that retained some replication ability. Draper, S. J. andHeeney, J. L. (2010). Viruses as vaccine vectors for infectious diseasesand cancer. Nat. Rev. 8: 62-71. Viral vectors studied includedherpesviruses. There is evidence suggesting that attenuatedreplication-competent viruses induce superior and more balanced immuneresponses against expressed antigens than replication-defective viruses.Peng, B. et al. (2005) Replicating rather than non-replicatingadenovirus-human immunodeficiency virus recombinant vaccines are betterat eliciting potent cellular immunity and priming high-titer antibodies.J. Virol. 79: 10200-9; Halford, W. P. et al. (2006) ICP0 antagonizesStat I-dependent repression of herpes simplex virus: implications forthe regulation of viral latency. Virol. J. 3: 44; Huang, X. et al.(2009) A novel replication-competent vaccinia vector MVTT is superior toMVA for inducing high levels of neutralizing antibody via mucosalvaccination. PLoS ONE 4: e4180. We hypothesize that thereplication-competent controlled viruses of the invention whichreplicate with near wild type efficiency will be similarly powerful oreven more powerful “adjuvants” than attenuated viruses.

There have been concerns ultimately relating to the effectiveness ofvirally vectored vaccines. One issue has been whether vaccine vectorscompete with transgene products for induction of immune responses.Paradoxically, strong immune responses to vector antigens may enhanceimmune responses to vector-expressed antigen(s) from another pathogen.Truckenmiller, M. E., Norbury, C. C. (2004) Viral vectors for inducingCD8 T cell responses. Exp. Opin. Biol. Ther. 4: 861-8. CD8 T cells canprovide help for other responding CD8+ cells if present in sufficientnumbers. Wang, B. et al. (2001) Multiple paths for activation of naïveCD8+ T cells: CD4-independent help. J. Immunol. 167: 1283-9.

Another concern has been whether pre-existing immunity to a virus willpreclude its use as a vaccine vector. This issue not only relates toviruses such as adenoviruses and herpes viruses that are endemic butalso to viruses that not normally infect humans but are used repeatedlyas vaccine vectors. There may have been more serious concerns regardingthe effects of pre-existing immunity to adenovirus (type 5) than to anyother vector. Draper, S. J., Heeney, J. L. (2010). A recent studydemonstrated that pre-existing immunity does not interfere with thegeneration of memory CD8 T cells upon vaccination with a heterologousantigen-expressing modified Ad5 vector, providing a basis for anefficient recall response and protection against subsequent challenge.Steffensen, M. A. et al. (2012) Pre-existing vector immunity does notprevent replication-deficient adenovirus from inducing efficient CD8T-cell memory and recall responses. PLoS ONE 7: e34884. Furthermore, thetransgene product-specific response could be boosted by re-vaccination.The issue of pre-existing immunity to herpesviruses has also beenexamined in multiple studies. Brockman, M. A. and Knipe, D. M. (2002)Herpes simplex virus vectors elicit durable immune responses in thepresence of preexisting host immunity. J. Virol. 76: 3678-87; Chahlavi,A. et al. (1999) Effect of prior exposure to herpes simplex virus 1 onviral vector-mediated tumor therapy in immunocompetent mice. Gene Ther.6: 1751-58; Delman, K. A. et al. (2000) Effects of preexisting immunityon the response to herpes simplex-based oncolytic therapy. Hum. GeneTher. 11: 2465-72; Hocknell, P. K. et al. (2002) Expression of humanimmunodeficiency virus type 1 gp120 from herpes simplex virus type1-derived amplicons result in potent, specific, and durable cellular andhumoral immune responses. J. Virol. 76: 5565-80; Lambright, E. S. et al.(2000) Effect of preexisting anti-herpes immunity on the efficacy ofherpes simplex viral therapy in a murine intraperitoneal tumor model.Mol. Ther. 2: 387-93; Herrlinger, U. et al. (1998) Pre-existing herpessimplex virus 1 (HSV-1) immunity decreases but does not abolish, genetransfer to experimental brain tumors by a HSV-1 vector. Gene Ther. 5:809-19; Lauterbach, H. et al. (2005) Reduced immune responses aftervaccination with a recombinant herpes simplex virus type 1 vector in thepresence of antiviral immunity. J. Gen. Virol. 86: 2401-10; Watanabe, D.et al. (2007) Properties of a herpes simplex virus multipleimmediate-early gene-deleted recombinant as a vaccine vector. Virology357: 186-98. A majority of these studies reported little effect or onlyrelatively minor effects on immune responses to herpesvirus-deliveredheterologous antigens or on anti-tumor efficacy of oncolyticherpesviruses. Brockman, M. A. and Knipe, D. M. (2002); Chahlavi, A. etal. (1999); Delman, K. A. et al. (2000); Hocknell, P. K. et al. (2002);Lambright, E. S. et al. (2000); Watanabe, D. et al. (2007). Two studieswere identified that reported substantial reductions of immuneresponses. Herrlinger, U. et al. (1998); Lauterbach, H. et al. (2005).However, it appears that the results of these studies may not begeneralized because compromised models were employed. One of the studiesemployed a tumor model that was only barely infectable with the mutantHSV strain used. Herrlinger, U. et al. (1998). The other study employeda chimeric mouse immune model in combination with a severely crippledHSV strain (ICP4⁻, ICP22⁻, ICP27⁻, vhs−) as the test vaccine.Lauterbach, H. et al. (2005). All studies agreed that vaccine uses ofherpesviruses are possible even in the presence of pre-existingimmunity. It may be added that pre-existing immunity, e.g., toherpesviruses, may not be a general issue for childhood vaccination.

A replication-competent controlled virus of the invention can also beemployed to control replication/propagation of a co-infecting secondimmunizing virus (or other microorganism). In the latter co-regulatedvirus, at least one replication-essential gene can be placed under thecontrol of a promoter that is responsive to the small-molecule-activatedtransactivator of the replication-competent controlled virus. To providean example, a co-regulated adenovirus may be constructed by replacingthe E1 and/or the E4 promoter with a transactivator-responsive promoter.Among viruses that could possibly be co-regulated in this fashion by areplication-competent controlled herpesvirus of the invention arepapillomaviruses (that also infect skin keratinocytes), certainpolyomaviruses (that infect skin fibroblasts and keratinocytes) andparvovirus B19 (fifth disease; infects skin fibroblasts). It is notedthat a co-regulated virus may naturally have an overlapping tropism withthe regulating virus. In certain cases it may also be possible togenerate a “matching tropism” by peudotyping. The group of viruses thatmay be co-regulated by a replication-competent controlled virus of theinvention may be expanded further and may even include RNA viruses ifco-regulation by means of regulated complementation is also taken intoconsideration. A pair of viruses (having overlapping tropisms) may,e.g., consist of a co-regulated virus that is defective for areplication-essential gene and a replication-competent controlled virusthat expresses the latter replication-essential gene under control of atransactivator-responsive promoter.

Viruses have evolved a multitude of mechanisms for evading immunedetection and avoiding destruction. Tortorella, D. et al. (2000) Viralsubversion of the immune system. Annu. Rev. Immunol. 18: 861-926.Elimination or weakening of some of these mechanisms could furtherenhance the immunogenicity of an immunizing virus or viral vector. Forexample, HSV-1 and HSV-2 express protein ICP47. This protein binds tothe cytoplasmic surfaces of both TAP1 and TAP2, the components of thetransporter associated with antigen processing TAP. Advani, S. J. andRoizman, B. (2005) The strategy of conquest. The interaction of herpessimplex virus with its host. In: Modulation of Host Gene Expression andInnate Immunity by Viruses (ed. P. Palese), pp. 141-61, Springer Verlag.ICP47 specifically interferes with MHC class I loading by binding to theantigen-binding site of TAP, competitively inhibiting antigenic peptidebinding. Virus-infected human cells are expected to be impaired in thepresentation of antigenic peptides in the MHC class I context and,consequently, to be resistant to killing by CD8+ CTL. Deletion ordisablement of the gene that encodes ICP47 ought to significantlyincrease the immunogenicity of the immunizing virus.

The role of ICP47 has been difficult to study in rodent models, becausethe protein is a far weaker inhibitor of mouse TAP than of human TAP.Still, one study was able to demonstrate that an HSV-1 ICP47-mutant wasless neurovirulent than the corresponding wild type strain and that thisreduced neurovirulence was due to a protective CD8 T cell response.Goldsmith, K. et al. (1998) Infected cell protein (ICP)47 enhancesherpes simplex virus neurovirulence by blocking the CD8+ T cellresponse. J. Exp. Med. 187: 341-8. Latently infected neurons may exhibitinfrequent but detectable expression of viral proteins. Feldman, L. T.et al. (2002) Spontaneous molecular reactivation of herpes simplex virustype 1 latency in mice. Proc. Natl. Acad. Sci. USA 99: 978-83. Theseproteins may be presented by MHC class I to specific CD8 T cells whoserole it may be to prevent virus reactivation. Khanna, K. M. et al.(2004) Immune control of herpes simplex virus during latency. Curr.Opin. Immunol. 16: 463-69. Co-localization of CD8 T cells with infectedcells in trigeminal ganglia has been observed. Khanna, K. M. et al.(2003) Herpes simplex virus-specific memory CD8 T cells are selectivelyactivated and retained in latently infected sensory ganglia. Immunity18: 593-603. That CD8 T cells control virus reactivation from latencyand that this control is dependent on MHC class I presentation wasdemonstrated in a mouse study using HSV-1 recombinants that expressedcytomegalovirus MHC class I inhibitors. Orr, M. T. et al. (2007) CD8 Tcell control of HSV reactivation from latency is abrogated by viralinhibition of MHC class I. Cell Host Microbe 2: 172-80. Hence, deletionof ICP47 is expected not only to enhance the immunogenicity of areplication-competent controlled virus but also to greatly reduce thealready low probability of its inadvertent reactivation from latency.

The immunogenicity of an immunizing virus (i.e., a replication-competentcontrolled virus of the invention) may also be enhanced by including inthe viral genome an expressible gene for a cytokine or other componentof the immune system. A vaccination study in mice in whichreplication-defective herpesvirus recombinants expressing variouscytokines were compared demonstrated that virus-expressed IL-4 and IL-2had adjuvant effects. Osiorio, Y., Ghiasi, H. (2003) Comparison ofadjuvant efficacy of herpes simplex virus type 1 recombinant virusesexpressing T_(H)1 and T_(H)2 cytokine genes. J. Virol. 77: 5774-83.Further afield, modulation of dendritic cell function by GM-CSF wasshown to enhance protective immunity induced by BCG and to overcomenon-responsiveness to a hepatitis B vaccine. Nambiar, J. K. et al.(2009) Modulation of pulmonary DC function by vaccine-encoded GM-CSFenhances protective immunity against Mycobacterium tuberculosisinfection. Eur. J. Immunol. 40: 153-61; Chou, H. Y. et al. (2010)Hydrogel-delivered GM-CSF overcomes nonresponsiveness to hepatitis Bvaccine through recruitment and activation of dendritic cells. J.Immunol. 185: 5468-75.

An effective amount of a replication-competent controlled virus of theinvention is an amount that upon administration to a subject and inducedreplication therein results in a detectably enhanced functional immunityof the subject (that is superior to the immunity induced by areplication-defective comparison virus). This enhanced functionalimmunity may manifest itself as enhanced resistance to infection orre-infection with a circulating (wild type) virus or may relate toenhanced suppression/elimination of a current infection. Hence, it mayalso manifest itself by a reduced disease severity, disease duration ormortality subsequent to infection with said wild type virus.Alternatively, or in addition, in the case of an immunizing virusexpressing a foreign antigen, immunity can relate to preventive ortherapeutic immunity against pathogens expressing and/or displaying thelatter foreign antigen. It is noted that a number of factors willinfluence what constitutes an effective amount of areplication-competent controlled virus, including to some extent thesite and route of administration of the virus to a subject as well asthe activation regimen utilized (i.e., the relative timing of heatingand small-molecule regulator administration, the heat dose(s) deliveredto the inoculation site region, the number of replicative cyclesinduced, etc.). Effective amounts of a replication-competent controlledvirus will be determined in dose-finding experiments. Generally, aneffective amount of a replication-competent controlled virus of theinvention will be from about 10² to about 10⁸ plaque-forming units (pfu)of virus. More preferably, an effective amount will be from about 10³ toabout 10⁷ pfu of virus, and even more preferably from about 10³ to about10⁶ pfu of virus. Conceivably, an effective amount of areplication-competent controlled virus of the invention may be outsideof the above ranges.

A vaccine composition of the invention will comprise an effective amountof a replication-competent controlled virus and, if a small-moleculeregulator is also administered as part of the composition, an effectiveamount of the small-molecule regulator. Although it may be administeredin the form of a fine powder under certain circumstances (as disclosed,e.g., in U.S. Pat. Appl. Publ. No 20080035143), a composition of theinvention typically is an aqueous solution comprising a virus of theinvention and, as the case may be, a small-molecule regulator. It may beadministered (parenterally) to a subject as an aqueous solution or, inthe case of administration to a mucosal membrane, as an aerosol thereof.See, e.g., U.S. Pat. No. 5,952,220. The compositions of the presentinvention will typically include a buffer component. The compositionswill have a pH that is compatible with the intended use, and istypically between about 6 and about 8. A variety of conventional buffersmay be employed such as phosphate, citrate, histidine, Tris, Bis-Tris,bicarbonate and the like and mixtures thereof. The concentration ofbuffer generally ranges from about 0.01 to about 0.25% w/v(weight/volume).

The compositions of the invention comprising a replication-competentcontrolled virus can further include, for example, preservatives, virusstabilizers, tonicity agents and/or viscosity-increasing substances. Asmentioned before, they may also include an appropriate small-moleculeregulator, or a formulation comprising such small-molecule regulator.

Preservatives used in parenteral products include phenol, benzylalcohol, methyl paraben/propylparaben and phenoxyethanol. Phenoxyethanolis the most widely used preservative found in vaccines. Preservativesare generally used in concentrations ranging from about 0.002 to about1% w/v. Meyer, B. K. 2007. Antimicrobial preservative use in parenteralproducts: past and present. J. Pharm. Sci. 96: 3155-67. Preservativesmay be present in compositions comprising a replication-competentcontrolled virus at concentrations at which they do not or onlyminimally interfere with the replicative efficiency of the virus.

Osmolarity can be adjusted with tonicity agents to a value that iscompatible with the intended use of the compositions. For example, theosmolarity may be adjusted to approximately the osmotic pressure ofnormal physiological fluids, which is approximately equivalent to about0.9% w/v of sodium chloride in water. Examples of suitable tonicityadjusting agents include, without limitation, chloride salts of sodium,potassium, calcium and magnesium, dextrose, glycerol, propylene glycol,mannitol, sorbitol and the like, and mixtures thereof. Preferably, thetonicity agent(s) will be employed in an amount to provide a finalosmotic value of 150 to 450 mOsm/kg, more preferably between about 220and about 350 mOsm/kg and most preferably between about 270 and about310 mOsm/kg.

If indicated, the compositions of the invention can further include oneor more viscosity-modifying agents such as cellulose polymers, includinghydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethylcellulose, hydroxypropyl cellulose, methyl cellulose, carboxymethylcellulose, glycerol, carbomers, polyvinyl alcohol, polyvinylpyrrolidone, alginates, carrageenans, guar, karaya, agarose, locust beangum, and tragacanth and xanthan gums. Such viscosity modifyingcomponents are typically employed in an amount effective to provide thedesired degree of thickening. Viscosity-modifying agents may be presentin compositions comprising a replication-competent controlled virus atconcentrations at which they do not or only minimally interfere withinfectivity and replicative efficiency of the virus.

If the composition also contains a small-molecule regulator, aneffective amount of such small-molecule regulator can be included in thecomposition in the form of a powder, solution, emulsion or particle. Asalso provided before, an effective amount of a small-molecule regulatorto be co-delivered with an effective amount of a replication-competentcontrolled virus will be an amount that yields an effectiveconcentration of small-molecule regulator in the inoculation siteregion, which effective concentration enables at least one round ofreplication of the replication-competent controlled virus in infectedcells of that region. To maintain a small-molecule regulator at aneffective concentration for a more extended period, i.e., if replicationof the virus (a heat- and small-molecule regulator-activated virus) isreinitiated by a second or further heat treatment of the inoculationsite region, the small-molecule regulator may be included in the form ofa slow-release formulation (see also below).

Methods for amplifying viruses are well known in the laboratory art.Industrial scale-up has also been achieved. For herpesviruses, seeHunter, W. D. (1999) Attenuated, replication-competent herpes simplexvirus type 1 mutant G207: safety evaluation of intracerebral injectionin nonhuman primates. J. Virol. 73: 6319-26; Rampling, R. et al. (2000)Toxicity evaluation of replication-competent herpes simplex virus(ICP34.5 null mutant 1716) in patients with recurrent malignant glioma.Gene Ther. 7: 859-866; Mundle, S. T. et al. (2013) High-puritypreparation of HSV-2 vaccine candidate ACAM529 is immunogenic andefficacious in vivo. PLoS ONE 8(2): e57224.

Various methods for purifying viruses have been disclosed. See, e.g.,Mundle et al. (2013) and references cited therein; Wolf, M. W. andReichl, U. (2011) Downstream processing of cell culture-derived virusparticles. Expert Rev. Vaccines 10: 1451-75.

While a small-molecule regulator can be co-administered with areplication-competent controlled virus in a single composition, acomposition comprising a replication-competent controlled virus and acomposition comprising a small-molecule regulator can also beadministered separately. The latter composition will comprise aneffective amount of a small-molecule regulator formulated together withone or more pharmaceutically acceptable carriers or excipients.

A composition comprising a small-molecule regulator may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The compositions maycontain any conventional non-toxic, pharmaceutically acceptablecarriers, adjuvants or vehicles. In some cases, the pH of theformulation may be adjusted with pharmaceutically acceptable acids,bases or buffers to enhance the stability of the formulatedsmall-molecule regulator or its delivery form. The term parenteral asused herein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

Liquid dosage forms of a small-molecule regulator for oraladministration include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In addition,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include, e.g., wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic, parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a small-molecule regulator, it may bedesirable to slow the absorption of the compound from, e.g.,subcutaneous (or, possibly, intracutaneous) or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the small-molecule regulator then depends upon its rateof dissolution, which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered small-molecule regulator is accomplished by dissolving orsuspending the compound in an oil vehicle. Injectable depot forms aremade by forming microcapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the small-moleculeregulator with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the small-moleculeregulator.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, thesmall-molecule regulator is mixed with at least one inert,pharmaceutically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and/or: a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid, b) binders suchas, for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the small-molecule regulator only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Dosage forms for topical or transdermal administration of asmall-molecule regulator include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants or patches. Thesmall-molecule regulator is admixed under sterile conditions with apharmaceutically acceptable carrier and any preservatives or buffers asmay be required.

The ointments, pastes, creams and gels may contain, in addition to asmall-molecule regulator, excipients such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the small-moleculeregulator, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons and replacements thereof.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin.

For pulmonary delivery, a composition comprising an effective amount ofa small-molecule regulator of the invention is formulated andadministered to the subject in solid or liquid particulate form bydirect administration e.g., inhalation into the respiratory system.Solid or liquid particulate forms of the small-molecule regulatorprepared for practicing the present invention include particles ofrespirable size: that is, particles of a size sufficiently small to passthrough the mouth and larynx upon inhalation and into the bronchi andalveoli of the lungs. Delivery of aerosolized therapeutics, particularlyaerosolized antibiotics, is known in the art (see, for example U.S. Pat.Nos. 5,767,068 and 5,508,269, and WO 98/43650). A discussion ofpulmonary delivery of antibiotics is also found in U.S. Pat. No.6,014,969.

What an effective amount of a small-molecule regulator is will depend onthe activity of the particular small-molecule regulator employed, theroute of administration, time of administration, the stability and rateof excretion of the particular small-molecule regulator as well as thenature of the specific composition administered. It may also depend onthe age, body weight, general health, sex and diet of the subject, otherdrugs used in combination or contemporaneously with the specificsmall-molecule regulator employed and like factors well known in themedical arts.

Ultimately, what is an effective amount of a small-molecule regulatorhas to be determined in dose-finding experiments, in which viralreplication is assessed experimentally in the inoculation site region.Once an effective amount has been determined in animal experiments, itmay be possible to estimate a human effective amount. “Guidance forIndustry. Estimating the maximum safe starting dose for initial clinicaltrials for therapeutics in adult healthy volunteers”, U.S. FDA, Centerfor Drug Evaluation and Research, July 2005, Pharmacology andToxicology. For example, as estimated from rat data, an effective humanamount of orally administered mifepristone (for enabling a single cycleof virus replication) will be between about 1 and about 100 μg/kg bodyweight.

If only a single round of replication of a replication-competentcontrolled virus of the invention is desired, small-molecule regulatormay be administered to a subject as a single dose. However, the sameamount may also be administered to a subject in divided doses. Asdiscussed before, if multiple rounds of virus replication are desired tobe induced within a relatively short period, an effective amount of asmall-molecule regulator may be administered (once) in a slow releaseformulation that is capable of sustaining an effective concentration ofsmall-molecule regulator for the period in question. Alternatively, aneffective amount of a small-molecule regulator (that is capable ofsustaining a single round of virus replication) can be administeredrepeatedly, whereby each administration is coordinated with the heatactivation of virus replication (in the case of a heat- andsmall-molecule regulator-activated virus).

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

The description herein of any aspect or embodiment of the inventionusing terms such as reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the invention that“consists of,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e. g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1: Construction of Replication-Competent ControlledViruses

All vectors were constructed using wild type HSV-1 strain 17syn+ as thebackbone. This strain is fully virulent, is well characterized, and thecomplete genomic sequence is available. The generation of the viralrecombinants was performed by homologous recombination of engineeredplasmids along with purified virion DNA into rabbit skin cells (RS) bythe calcium phosphate precipitation method as previously described.Bloom, D. C. 1998. HSV Vectors for Gene Therapy. Methods Mol. Med. 10:369-386. All plasmids used to engineer the insertions of thetransactivators or the GAL4-responsive promoters for recombination intothe HSV-1 genome were cloned from HSV-1 strain 17syn+. Plasmid IN994 wascreated as follows: an HSV-1 upstream recombination arm was generated byamplification of HSV-1 DNA (17+) (from base pairs 95,441 to 96,090) withDB112 (5′GAG CTC ATC ACC GCA GGC GAG TCT CTT3′) and DB113 (5′GAG CTC GGTCTT CGG GAC TAA TGC CTT3′). The product was digested with SacI andinserted into the SacI restriction site of pBluescript to create pUP. AnHSV-1 downstream recombination arm was generated using primersDB115-KpnI (5′GGG GTA CCG GTT TTG TTT TGT GTG AC3′) and DB120-KpnI(5′GGG GTA CCG GTG TGT GAT GAT TTC GC3′) to amplify HSV-1 (17+ strain)genomic DNA sequence between base pairs 96,092 and 96,538. The PCRproduct was digested with KpnI, and cloned into KpnI digested pUP tocreate pIN994, which recombines with HSV-1 at the intergenic UL43/44region.

HSV-GS1 contains a transactivator (TA) gene cassette inserted into theintergenic region between UL43 and UL44. In addition, the ICP4 promoterhas been replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats. Afirst recombination plasmid pIN:TA1 was constructed by inserting a DNAsegment containing a glp65 gene under the control of a promoter cassettethat combined a human hsp70B promoter and a GAL4-responsive promoter(described in Vilaboa et al. 2005) into the multiple cloning site ofplasmid pIN994, between flanking sequences of the HSV-1 UL43 and UL44genes. The TA cassette was isolated from plasmid Hsp70/GAL4-GLP65(Vilaboa et al. 2005) and was cloned by 3-piece ligation to minimize theregion that was amplified by PCR. For the left insert, Hsp70/GAL4-GLP65was digested with BamHI and BstX1 and the resulting 2875 bp band was gelpurified. This fragment contains the Hsp70/GAL4 promoter cassette aswell as the GAL4 DNA-binding domain, the progesterone receptorligand-binding domain and part of the p65 activation domain oftransactivator GLP65. The right insert was generated by amplifying aportion of pHsp70/GAL4-GLP65 with the primers TA.2803-2823.fwd (5′TCGACA ACT CCG AGT TTC AGC3′) (SEQ ID NO: 1) and BGHpA.rev (5′ CTC CTC GCGGCC GCA TCG ATC CAT AGA GCC CAC CGC ATC C3′) (SEQ ID NO: 2). The 763 bpPCR product was digested with BstX1 and NotI, and the resultant 676 bpband was gel-purified. This band contained the 3′end of the p65activation domain and the BGHpA. For the vector, pIN994 was digestedwith BamHI and NotI, and the resulting 4099 bp fragment was gel-purifiedand shrimp alkaline phosphatase (SAP)-treated. The two inserts were thensimultaneously ligated into the vector, creating an intact TA cassette.Subsequent to transformation, colony #14 was expanded, and the plasmidwas verified by restriction enzyme analysis and then by sequenceanalysis.

One μg of pIN:TA1 was co-transfected with 2 μg of purified HSV-1 (17+)virion DNA into RS cells by calcium phosphate precipitation. Theresulting pool of viruses was screened for recombinants by pickingplaques, amplifying these plaques on 96 well plates of RS cells, anddot-blot hybridization with a ³²P-labeled DNA probe prepared by labelinga TA fragment by random-hexamer priming. A positive well was re-plaquedand re-probed 5 times and verified to contain the TA by PCR and sequenceanalysis. This intermediate recombinant was designated HSV-17GS43.

A second recombination plasmid, pBS-KS:GAL4-ICP4, was constructed thatcontained a GAL4-responsive promoter inserted in place of the nativeICP4 promoter by cloning it in between the HSV-1 ICP4 recombination armsin the plasmid pBS-KS:ICP4Δpromoter. This placed the ICP4 transcriptunder the control of the exogenous GAL4 promoter. This particularpromoter cassette includes six copies of the yeast GAL4 UAS (upstreamactivating sequence), the adenovirus E1b TATA sequence and the syntheticintron Ivs8. This cassette was excised from the plasmid pGene/v5-HisA(Invitrogen Corp.) with AatII and HindIII, and the resulting 473 bpfragment was gel-purified. For the vector, pBS-KS:ICP4Δpromoter wasdigested with AatII and HindIII and the resulting 3962 bp fragmentgel-purified and SAP-treated. Ligation of these two fragments placed theGAL4 promoter in front of the ICP4 transcriptional start-site.Subsequent to transformation, colony #5 was expanded, test-digested andverified by sequencing.

One μg of pBS-KS:GAL4-ICP4 was co-transfected with 4 μg of purifiedHSV-17GS43 virion DNA into cells of the ICP4-complementing cell line E5(DeLuca, N. A. and Schaffer, P. A. 1987. Activities of herpes simplexvirus type 1 (HSV-1) ICP4 genes specifying nonsense peptides. NucleicAcids Res. 15: 4491-4511) by calcium phosphate precipitation. Theresulting pool of viruses was screened for recombinants by pickingplaques, amplifying these plaques on 96 well plates of E5 cells, anddot-blot hybridization with a ³²P-labeled DNA probe prepared by labelingthe GAL4-responsive promoter fragment by random-hexamer priming. Apositive well was re-plaqued and re-probed 7 times and verified tocontain the GAL4-responsive promoter in both copies of the short repeatsequences by PCR and sequence analysis. This recombinant was designatedHSV-GS1.

To obtain pBS-KS:ΔSacI, the SacI site was deleted from the polylinker ofplasmid vector pBluescript-KS+, by digesting the plasmid with SacI. Theresulting 2954 bp fragment was gel-purified, treated with T4 DNApolymerase to produce blunt ends, re-circularized and self-ligated.Recombination plasmid BS-KS:ICP4Δpromoter was constructed as follows: togenerate a first insert, cosmid COS48 (a gift of L. Feldman) wassubjected to PCR with the primers HSV1.131428-131404 (5′ CTC CTC AAG CTTCTC GAG CAC ACG GAG CGC GGC TGC CGA CAC G3′) (SEQ ID NO: 3) andHSV1.130859-130880 (5′ CTC CTC GGT ACC CCA TGG AGG CCA GCA GAG CCA GC3′)(SEQ ID NO: 4). The primers placed HindIII and XhoI sites on the 5′ endof the region and NcoI and KpnI sites on the 3′ end, respectively. The600 bp primary PCR product was digested with HindIII and KpnI, and theresulting 587 bp fragment was gel-purified. Vector pBS-KS:Δ.SacI wasdigested with HindIII and KpnI, and the resulting 2914 bp fragment wasgel-purified and SAP-treated. Ligation placed the first insert into thevector's polylinker, creating pBS-KS:ICP4-3′end. To generate a secondinsert, cosmid COS48 was subjected to PCR with the primersHSV1.132271-132250 (5′ CTC CTC GCG GCC GCA CTA GTT CCG CGT GTC CCT TTCCGA TGC3′) (SEQ ID NO: 5) and HSV1.131779-131800 (5′ CTC CTC CTC GAG AAGCTT ATG CAT GAG CTC GAC GTC TCG GCG GTA ATG AGA TAC GAG C3′) (SEQ ID NO:6). These primers placed NotI and SpeI sites on the 5′ end of the regionand AatII, SacI, NsiI, HindIII and XhoI sites on the 3′ end,respectively. The 549 bp primary PCR product was digested with NotI andXhoI, and the resulting 530 bp band was gel-purified. This fragment alsocontained the 45 bp OriS hairpin. Plasmid BS-KS:ICP4-3′ end was digestedwith NotI and XhoI and the resulting 3446 bp band was gel-purified andSAP-treated. Ligation generated pBS-KS:ICP4Δpromoter. The inserts inpBS-KS:ICP4Δpromoter were verified by sequence analysis.

HSV-GS2 contains transactivator (TA) gene cassette inserted into theintergenic region between UL37 and UL38. In addition, the ICP4 promoterhas been replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats. Arecombination plasmid, pUL37/38:TA, was constructed by inserting a DNAsegment containing a glp65 gene under the control of a promoter cassettethat combined a human hsp70B promoter and a GAL4-responsive promoterinto the BspE1/AfIII site of plasmid pBS-KS:UL37/38, between flankingsequences of the HSV-1 UL37 and UL38 genes. The TA cassette was isolatedfrom plasmid Hsp70/GAL4-GLP65 (Vilaboa et al. 2005) and was cloned by3-piece ligation to minimize the region that was amplified by PCR. Forthe left insert, pHsp70/GAL4-GLP65 was digested with BamHI (filled in)and BstX1 and the resulting 2875 bp band was gel-purified. This fragmentcontains the Hsp70/GAL4 promoter cassette as well as the GAL4 DNAbinding domain, the progesterone receptor ligand-binding domain and partof the p65 activation domain of transactivator GLP65. The right insertwas generated by amplifying a portion of pHsp70/GAL4-GLP65 with theprimers TA.2803-2823.fwd and BGHpA.rev. The 763 bp PCR product wasdigested with BstX1 and NotI (filled in), and the resultant 676 bp bandwas gel-purified. This band contained the 3′end of the p65 activationdomain and the BGHpA. For the vector, pBS-KS:UL37/38 was digested withBspE1 and AflII, and the resulting 3,772 bp fragment was filled in withT4 DNA polymerase, gel-purified and SAP-treated. The two inserts werethen simultaneously ligated into the vector, creating an intact TAcassette. Following transformation, colonies were screened byrestriction digestion. Colony #29 was expanded, and the plasmid verifiedby restriction enzyme analysis and then by sequence analysis.

One μg of pUL37/38:TA was co-transfected with 2 μg of purified HSV-1(17+) virion DNA into RS cells by calcium phosphate precipitation. Theresulting pool of viruses was screened for recombinants by pickingplaques, amplifying these plaques on 96 well plates of RS cells, anddot-blot hybridization with a ³²P-labeled DNA probe prepared by labelinga TA fragment by random-hexamer priming. A positive well was re-plaguedand re-probed 6 times and verified to contain the TA by PCR and sequenceanalysis. This intermediate recombinant was designated HSV-17GS38.

One μg of pBS-KS:GAL4-ICP4 was co-transfected with 5 μg of purifiedHSV-17GS38 virion DNA into E5 cells by calcium phosphate precipitation.The resulting pool of viruses were screened for recombinants by pickingplaques, amplifying these plaques on 96 well plates of E5 cells, anddot-blot hybridization with a ³²P-labeled DNA probe prepared by labelingthe GAL4-responsive promoter fragment by random-hexamer priming. Apositive well was re-plaqued and re-probed 7 times and verified tocontain the GAL4-responsive promoter in both copies of the short repeatsequences by PCR and sequence analysis. This recombinant was designatedHSV-GS2.

HSV-GS3 contains a transactivator (TA) gene cassette inserted into theintergenic region between UL43 and UL44. In addition, the ICP4 promoterhas been replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats.Furthermore, the ICP8 promoter was replaced with a GAL4-responsivepromoter. The construction of this recombinant virus involved placing asecond HSV-1 replication-essential gene (ICP8) under control of aGAL4-responsive promoter. HSV-GS1 was used as the “backbone” for theconstruction of this recombinant. ICP8 recombination plasmidpBS-KS:GAL4-ICP8 was constructed. This plasmid contained aGAL4-responsive promoter inserted in place of the native ICP8 promoterby cloning it in between the HSV-1 ICP8 recombination arms in theplasmid pBS-KS:ICP8Δpromoter. This placed the ICP8 transcript under thecontrol of the exogenous GAL4-responsive promoter. This particularpromoter cassette consisted of six copies of the yeast GAL4 UAS(upstream activating sequence), the adenovirus E1 b TATA sequence andthe synthetic intron Ivs8. This cassette was excised from the plasmidpGene/v5-HisA (Invitrogen Corp.) with AatII and HindIII, and theresulting 473 bp fragment gel-purified. For the vector,pBS-KS:ICP8Δpromoter was digested with AatII and HindIII, and theresulting 4588 bp fragment gel-purified and SAP-treated. Ligation of thelatter two DNA fragments placed the GAL4-responsive promoter cassette infront of the ICP8 transcriptional start-site. Subsequent totransformation, colony #10 was expanded, test-digested and verified bysequencing.

One μg of pBS-KS:GAL4-ICP8 was co-transfected with 10 μg of purifiedHSV-GS1 virion DNA into E5 cells by calcium phosphate precipitation.Subsequent to the addition of mifepristone to the medium, thetransfected cells were exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently on days 2 and 3, the cells were againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaqueswere picked and amplified on 96 well plates of E5 cells in mediasupplemented with mifepristone. The plates were incubated at 43.5° C.for 30 minutes 1 hour after infection and then incubated at 37° C.Subsequently on days 2 and 3, the plates were also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells showed90-100% CPE, the plates were dot-blotted and the dot-blot membranehybridized with a ³²P-labeled DNA probe prepared by labeling the HSV-1ICP8 promoter fragment that was deleted. A faintly positive well wasre-plaqued and re-probed 8 times and verified to have lost the ICP8promoter and to contain the GAL4-responsive promoter in its place by PCRand sequence analysis. This recombinant was designated HSV-GS3.

Recombination plasmid pBS-KS:ICP8Δpromoter was constructed usingessentially the same strategy as that described above for the creationof pBS-KS:ICP4Δpromoter: a first insert was PCR-amplified from HSV-117syn+ virion DNA using the primers HSV1.61841-61865 (5′ CTC CTC AGA ACCCAG GAC CAG GGC CAC GTT GG3′) (SEQ ID NO: 7) and HSV1.62053-62027 (5′CTC CTC ATG GAG ACA AAG CCC AAG ACG GCA ACC3′) (SEQ ID NO: 8) andsubcloned to yield intermediate vector pBS-KS:ICP8-3′ end. A secondinsert was similarly obtained using primers HSV1.62173-62203 (5′ CTC CTCGGA GAC CGG GGT TGG GGA ATG AAT CCC TCC3′) (SEQ ID NO: 9) andHSV1.62395-62366 (5′ CTC CTC GCG GGG CGT GGG AGG GGC TGG GGC GGA CC3′)(SEQ ID NO: 10) and was subcloned into pBS-KS:ICP8-3′ end to yieldpBS-KS:ICP8Δpromoter.

HSV-GS4: contains a transactivator (TA) gene cassette inserted into theintergenic region between UL43 and UL44. In addition, the ICP4 promoterhas been replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats,and the ICP8 promoter has been replaced with a GAL4-responsive promoter.Furthermore, the US12 gene has been mutated to render its proteinproduct (ICP47) nonfunctional. ICP47 amino acid residue K31 was changedto G31, and R32 to G32. Neumann, L. et al. (1997) J. Mol. Biol. 272:484-92; Galocha, B. et al. (1997) J. Exp. Med. 185: 1565-72. A 500 bpICP47 coding sequence-containing fragment was PCR-amplified from virionDNA of strain 17syn+. The fragment was PCR-amplified as two pieces (a“left-hand” and a “right-hand” piece), using two primer pairs. Themutations were introduced through the 5′ PCR primer for the right-handfragment. The resulting amplified left-hand and mutated right-handfragments were subcloned into vector pBS, and the sequence in subcloneswas confirmed by sequence analysis. A subclone containing the 500 bpfragment with the desired mutations in ICP47 codons 31 and 32 was termedpBS:mut-ICP47.

One μg of pBS:mut-ICP47 was co-transfected with 10 μg of purifiedHSV-GS3 virion DNA into E5 cells by calcium phosphate precipitation.Subsequent to the addition of mifepristone to the medium, thetransfected cells were exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently on days 2 and 3, the cells were againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaqueswere picked and amplified on 96 well plates of E5 cells in mediasupplemented with mifepristone. The plates were incubated at 43.5° C.for 30 minutes 1 hour after infection and then incubated at 37° C.Subsequently on days 2 and 3, the plates were also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells showed90-100% CPE, the plates were dot-blotted and the dot-blot membranehybridized with a ³²P-labeled oligonucleotide probe to the mutated ICP47region. A positive well was re-plagued and re-probed several times andverified by sequence analysis to contain the expected mutated ICP47 genesequence. This recombinant was designated HSV-GS4.

HSV-GS5 contains an expressible (auto-activated) transactivator (TA)gene inserted into the intergenic region between UL43 and UL44. Inaddition, the ICP4 promoter has been replaced with a GAL4-responsivepromoter (GAL4-binding site-containing minimal promoter) in both copiesof the short repeats. A recombination plasmid pIN:TA2 is constructed byinserting a DNA segment containing an auto-activated glp65 gene into themultiple cloning site of plasmid pIN994, between flanking sequences ofthe HSV-1 UL43 and UL44 genes. The expressible TA gene is isolated frompHsp70/GAL4-GLP65 (Vilaboa et al. 2005) and pSwitch (Invitrogen lifetechnologies), respectfully, and is cloned by 3-piece ligation tominimize the region that is amplified by PCR. For the left insert,pSwitch is digested with Ssp1 and BstX1, and a resulting 2425 bp band isgel purified. This fragment contains the auto-activated promoter as wellas the GAL 4 DNA-binding domain, the progesterone receptorligand-binding domain and part of the p65 activation domain oftransactivator GLP65. The right insert is generated by amplifying aportion of pHsp70/GAL4-GLP65 with the primers TA.2803-2823.fwd andBGHpA.rev. The 763 bp PCR product is digested with BstX1 and NotI, andthe resultant 676 bp band is gel-purified. This band contains the 3′endof the p65 activation domain and the BGHpA. For the vector, pIN994 isfirst digested with BamHI, ends are filled in with Klenow DNApolymerase, and the DNA is further digested with NotI. The resulting4099 bp fragment is gel-purified. The two inserts are thensimultaneously ligated into the vector, creating an intact expressibleTA gene. Subsequent to transformation, several colonies are expanded andplasmid DNAs subjected to restriction and then sequence analysis toidentify pIN:TA2.

One μg of pIN:TA2 is co-transfected with 2 μg of purified HSV-1 virionDNA into RS cells by calcium phosphate precipitation. The resulting poolof viruses is screened for recombinants by picking plaques, amplifyingthese plaques on 96 well plates of RS cells, and dot-blot hybridizationwith a ³²P-labeled DNA probe prepared by labeling a TA fragment byrandom-hexamer priming. A positive well is re-plaqued and re-probedseveral times and verified to contain the TA by PCR and sequenceanalysis. This intermediate recombinant is designated HSV-17GS43A.

One μg of pBS-KS:GAL4-ICP4 is co-transfected with 4 μg of purifiedHSV-17GS43A virion DNA into cells of the ICP4-complementing cell line E5by calcium phosphate precipitation. The resulting pool of viruses isscreened for recombinants by picking plaques, amplifying these plaqueson 96 well plates of E5 cells, and dot-blot hybridization with a³²P-labeled DNA probe prepared by labeling the GAL4-responsive promoterfragment by random-hexamer priming. A positive well is re-plaqued andre-probed several times and verified to contain the GAL4-responsivepromoter in both copies of the short repeat sequences by PCR andsequence analysis. This recombinant is designated HSV-GS5.

HSV-GS6 contains an auto-activated transactivator (TA) gene insertedinto the intergenic region between UL43 and UL44. In addition, the ICP4promoter is replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats.Furthermore, the ICP8 promoter is replaced with a human hsp70B promoter.The construction of this recombinant virus involves placing a secondHSV-1 replication-essential gene (ICP8) under control of an Hsp70Bpromoter. HSV-GS5 is used as the “backbone” for the construction of thisrecombinant. ICP8 recombination plasmid pBS-KS:Hsp70B-ICP8 isconstructed that contains an hsp70B promoter inserted in place of thenative ICP8 promoter by cloning it in between the HSV-1 ICP8recombination arms in the plasmid pBS-KS:ICP8Δpromoter. To isolate ahuman hsp70B promoter fragment, construct p17 is digested with BamHI,ends are filled in by Klenow DNA polymerase, and the DNA is furtherdigested with HindIII. A 450 bp promoter fragment is gel-purified(Voellmy, R. et al. (1985) Proc. Natl. Acad. Sci. USA 82: 4949-53). Forthe vector, pBS-KS:ICP8Δpromoter is digested with ZraI and HindIII. Theresulting 4588 bp fragment is gel-purified. Ligation of the latter twoDNA fragments places the hsp70B promoter in front of the ICP8transcriptional start-site. Subsequent to transformation, severalcolonies are expanded and plasmid DNAs subjected to restriction and thensequence analysis to identify pBS-KS:Hsp70B-ICP8.

One μg of pBS-KS:Hsp70B-ICP8 is co-transfected with 10 μg of purifiedHSV-GS5 virion DNA into E5 cells by calcium phosphate precipitation. Thetransfected cells are exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently on days 2 and 3, the cells are againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaquesare picked and amplified on 96 well plates of E5 cells. The plates areincubated at 43.5° C. for 30 minutes a few hours after infection andthen incubated at 37° C. Subsequently on days 2 and 3, the plates arealso shifted to 43.5° C. for 30 minutes and then returned to 37° C.After the wells show 90-100% CPE, the plates are dot-blotted and thedot-blot membrane hybridized with a ³²P-labeled DNA probe prepared bylabeling the hsp70B promoter fragment by random-hexamer priming. Apositive well is re-plaqued and re-probed several times and verified tohave lost the ICP8 promoter and to contain the hsp70B promoter in itsplace by PCR and sequence analysis. This recombinant is designatedHSV-GS6.

Generally known molecular biology and biochemistry methods were used.Molecular biology methods are described, e.g., in “Current protocols inmolecular biology”, Ausubel, F. M. et al., eds., John Wiley and Sons,Inc. ISBN: 978-0-471-50338-5.

Example 2: Regulated Replication of the Replication-Competent ControlledViruses

(a) Growth and Replication Properties of HSV-GS1

Plaque Analysis on Permissive (E5) and Non-Permissive (RS) Cells

Serial dilutions of the purified HSV-GS1 virus were plated ontoconfluent monolayers of either rabbit skin (RS) or ICP4-complementingVero-based helper cell line (E5) cells in 60 mm dishes. The virus wasallowed to adsorb for 1 hour at 37° C., and then the inoculum wasremoved, and the cells were overlayed with complete medium (ModifiedEagles Medium supplemented with 5% calf serum or 10% fetal bovine serumfor RS or E5 cells, respectively). The dishes were then incubated 72hours and stained with crystal violet to visualize any viral plaques. Atdilutions resulting in 10-100 plaques on E5 cells, no plaques wereobserved on the non-complementing RS cells.

Growth Analysis of HSV-GS1

Confluent monolayers of either RS or E5 cells in 60 mm dishes wereinfected as described above with HSV-GS1 at a multiplicity-of-infection(m.o.i.) of 5, incubated for 48 hours, harvested by scraping cells intothe medium, frozen at −80° C., subjected to 2 rounds of freezing-thawingand titrated for infectious virus. Results are shown in Table 1.

TABLE 1 Titration of viruses in RS and E5 cells Virus RS cells E5 cells17syn+ 8.3 × 10⁸ pfu/ml 1.2 × 10^(7 pfu/ml) HSV-GS1 ND* 4.0 × 10⁷ pfu/ml*ND = None detected. (Detection limit in this experiment was about 100PFU.)Analysis of the Effects of Heat Exposure and Mifepristone on theReplication of HSV-GS1 in Vero Cells

The purpose of this experiment was to compare the replication cycle ofHSV-GS1 with the wild type vector HSV-1 strain 17syn+. Confluentmonolayers of Vero cells were infected with either HSV-1 strain 17+ orthe recombinant HSV-GS1 at an m.o.i. of 3. The virus was allowed toadsorb for 1 hour at 37° C., and then the inoculum was removed, and thecells were overlayed with complete medium (Modified Eagles Mediumsupplemented with 10% fetal bovine serum). Heat treatment was performed4 hours after adsorption by floating the sealed dishes in a 43.5° C.water bath for 30 minutes. Mifepristone treatment (10 nM) was initiatedat the time of the initial infection. The dishes were then incubated for72 hours at 37° C. At 0, 8, 16 and 28 hours post-infection, two disheswere removed, the cells scraped into the media to harvest, and subjectedto 2 freeze-thaw cycles. The infectious virus was then determined bytitrating the lysate of each dish in triplicate on 24-well plates ofconfluent E5 cells. Plaques were visualized after 2 days by stainingwith crystal violet. Results demonstrate that under the chosenexperimental conditions HSV-GS1 replicates as efficiently as wild typevirus 17syn+(FIG. 3B). No replication of HSV-GS1 appears to occur in theabsence of an activation treatment (heat and mifepristone). It is alsonoted that the activating treatment, i.e., heat exposure and incubationin the presence of mifepristone, only slightly affected wild type virusreplication.

Analysis of the Dependence of HSV-GS1 Replication on Both Heat Exposureof the Host Cell and the Presence of Small-Molecule Regulator

The purpose of this experiment was to determine whether the activationof the HSV-GS1 recombinant by heat and mifepristone was due to arequirement of both, and that mifepristone alone or heat alone was notsufficient to induce replication. The experiment was performed asdescribed in the preceding section with the exception that heattreatment was administered immediately after adsorption. The data showthat replication of HSV-GS1 did not occur unless the host cells wereexposed to heat in the presence of mifepristone (FIG. 3A).

(b) Growth and Replication Properties of HSV-GS3

Replication Efficiency of HSV-GS3

The purpose of this experiment was to compare the replication cycle ofthe HSV-GS3 recombinant with the wild type vector HSV-1 17syn+.Confluent monolayers of E5 cells (but see below) were infected witheither HSV-1 strain 17+ or the recombinant HSV-GS3 at an m.o.i. of 3.Virus was allowed to adsorb for 1 hour at 37° C. and then the inoculumwas removed and the cells overlayed with complete medium (ModifiedEagles Medium supplemented with 10% fetal bovine serum). Heat treatmentwas performed after adsorption by floating the sealed dishes in a 43.5 Cwater bath for 30 minutes at 4 hours post infection. Mifepristonetreatment (10 nM) was initiated at the time of the initial infection.For the HSV-GS3 No Tx (no treatment) set, the E5 cells were transfectedwith a plasmid containing an expressible ICP8 gene 12 hours prior toinfection. The dishes were then incubated further at 37° C. At 0, 4, 12and 24 hours post-infection, two dishes were removed, the cells scrapedinto the media to harvest, and subjected to 2 freeze-thaw cycles. Theinfectious virus was then determined by titrating the lysate of eachdish in triplicate on 24-well plates of confluent E5 cells previouslytransfected with ICP8 expression plasmid. Plaques were visualized after2 days by staining with crystal violet. Results indicate that HSV-GS3replicated nearly as efficiently as wild-type virus HSV-1 17syn+ underthe chosen experimental conditions (FIG. 4A).

Analysis of the Dependence of HSV-GS3 Replication on Both Heat Exposureof the Host Cell and the Presence of Small-Molecule Regulator

Single-step growth experiments were carried out to determine whether theactivation of HSV-GS3 by heat and mifepristone was due to a requirementof both, and that mifepristone alone or heat alone was not sufficient toinduce replication. The experiment shown in FIG. 4B was performed asdescribed in the preceding section with the exception that Vero cellswere used and heat treatment was administered immediately afteradsorption. Titrations were in E5 cells previously transfected with ICP8expression plasmid. A similar experiment was carried out with humansquamous cell tumor line SCC-15 (FIG. 4C). The results of the latterexperiments demonstrate that replication of HSV-GS3 is tightlyregulated. It is only triggered by heat exposure of infected cells inthe presence of mifepristone.

In other experiments, measurement of virus replication by titration ofinfectious virus was substituted by methods of quantification of viralDNA or expression of viral genes. In these experiments mifepristoneand/or ulipristal (small-molecule regulators) were tested. One suchexperiment had the design summarized in Table 2.

TABLE 2 Treatment groups Ulipristal Mifepristone No drug 0.1 nM 0.3 nM 1nM 10 nM 10 nM — Heat X X X X X X treatment No heat X X X treatment

Thirty-five mm dishes of confluent Vero cells were infected at an m.o.i.of 3 with the HSV-GS3 vector. Each treatment group consisted of 3replicate dishes (for each time point). Virus was adsorbed for 1 hour at37° C., the inoculum removed, and the cells overlayed with completemedium (Modified Eagles Medium supplemented with 10% fetal bovineserum). Drug (i.e., mifepristone or ulipristal) treatment was initiatedat the time of the initial infection. Heat treatment was performed afteradsorption by floating the sealed dishes in a 43.5° C. water bath for 30minutes on a submerged platform (initiated 4 hours post infection).Dishes were incubated further at 37° C. At 1, 4, 12 and 24 h post heattreatment, three dishes were removed, the media removed, and the DNA andRNA extracted using TRIzol. Extracted DNA was subjected to TaqmanRealtime PCR for quantitative analysis for HSV-1 DNA (using HSV DNApolymerase primers/probe). Extracted RNA was analyzed by Taqman RT-PCRfor the presence of ICP4 and glycoprotein C (gC) transcripts. DNA andRNA quantities were normalized relative to the cellular gene APRT andpresented as relative quantities. Primers used are shown in Table 3below.

TABLE 3 Primers and probes used for qPCR HSV DNA FAGAGGGACATCCAGGACTTTGT (SEQ ID NO: 11) Pol RCAGGCGCTTGTTGGTGTAC(SEQ ID NO: 12) P ACCGCCGAACTGAGCA (SEQ ID NO: 13)ICP4 F CACGGGCCGCTTCAC (SEQ ID NO: 14) RGCGATAGCGCGCGTAGA (SEQ ID NO: 15) P CCGACGCGACCTCC (SEQ ID NO: 16) gC FCCTCCACGCCCAAAAGC (SEQ ID NO: 17) RGGTGGTGTTGTTCTTGGGTTTG (SEQ ID NO: 18) P CCCCACGTCCACCCC (SEQ ID NO: 19)Mouse F CTCAAGAAATCTAACCCCTGACTCA  APRT (SEQ ID NO: 20) RGCGGGACAGGCTGAGA (SEQ ID NO: 21) P CCCCACACACACCTC (SEQ ID NO: 22) F:forward; R: reverse; P: probe.

Results obtained are shown in FIG. 5. FIG. 5A shows that viral DNAreplication depended both on heat treatment and small-moleculeregulator. For ulipristal it was demonstrated that DNA replication wasdependent on regulator dose. Compatible data were obtained for theexpression of the ICP4 genes (regulated genes) and the late gC gene (notsubject to deliberate regulation) (FIGS. 5B & C).

Example 3: Apparent Inability of a Replication-Competent ControlledVirus to Replicate In Vivo in the Absence of Deliberate Activation

The goal of this experiment was to demonstrate that, in the absence ofheat and small-molecule regulator, the GS vectors are as tightly “off”in mice as they appear to be in cell culture. Four to six week oldout-bred ND4 Swiss Webster female mice (Harlan Sprague-Dawley, Inc.)were infected with similar amounts of HSV-GS1 or 17syn+ wild type viruson the lightly abraded plantar surface of both rear feet followingsaline pre-treatment. At 4, 8, and 21 days post infection, 4 mice pertime point were euthanized and the feet and dorsal root ganglia (DRG)were harvested, homogenized in TRIzol, and DNA and RNA extracted. DNAwas subjected to Taqman Realtime PCR for quantitative analysis for HSV-1DNA; RNA was analyzed following RT by Taqman Realtime PCR for thepresence of ICP4 and glycoprotein C (gC) transcripts as previouslydescribed. Kubat, N.J. et al. 2004. The herpes simplex virus type 1latency-associated transcript (LAT) enhancer/rcr is hyperacetylatedduring latency independently of LAT transcription. J. Virol. 78:12508-18. The real-time primer/probe sequences are disclosed in Table 3.

Results showed a complete absence of HSV-GS1 gene expression in feet aswell as in DRG (Table 4). Consistent with this result implying thatHSV-GS1 was incapable of replication was the finding that DNA amounts ofHSV-GS1 were orders of magnitude lower than those of wild-type virus HSV17syn+. Replication efficiency difference at 8 days was 151 fold in feetand 200 fold in DRG, respectively.

TABLE 4 HSV-GS1 and wild type HSV-1: replication and viral geneexpression HSV 17+ HSV-GS1 Time post RNA RNA infection DNA ICP4 gC DNAICP4 gC Feet: 4 49,500 1,290 5,742 1,222 ND ND 8 35,773 976 5,332 237 NDND 21 49 ND* ND ND ND ND DRG: 4 12,674 576 3,563 59 ND ND 8 14,986 8779,230 75 ND ND 21 5,754 ND* ND 45 ND ND *ND = below the limit ofdetection.

Example 4: Activation In Vivo of a Replication-Competent ControlledVirus of the Invention

In these experiments, virus replication in a mouse model was estimatedby the biochemical methods of quantification of viral DNA and expressionof viral genes. DNA amounts and viral transcript amounts were measuredat both the foot (site of virus inoculation) and in the dorsal rootganglia (DRG) (site of HSV acute replication and latency). One suchexperiment had the design summarized in Table 5. This experiment wasalso aimed at determining the lowest effective in vivo dose ofulipristal.

TABLE 5 Treatment groups Ulipristal No drug 1 μg/kg 5 μg/kg 50 μg/kg —Heat X X X X treatment No heat X treatment

Outbred Swiss-webster female mice (4-6 weeks old) were inoculated with1×10⁵ pfu of HSV-GS3 vector following saline-pretreatment and lightabrasion of both rear footpads. Each treatment group consisted of 5mice. Drug treatment (ulipristal) was administered IP at the time ofinfection. Heat treatment was performed at 45° C. for 10 min (byimmersion of hind feet in a waterbath) 3 h after virus administration.Mice were allowed to recover at 37° C. for 15 min. Mice were sacrificed24 hours post heat induction, and the feet and DRG were dissected andsnap-frozen in RNA/ater (Sigma-Aldrich). DNA and RNA were extracted bygrinding the tissues in TRIzol (Life Technologies), and back-extractingthe DNA from the interface. DNA was subjected to Taqman Realtime PCR forquantitative analysis for HSV-1 DNA. RNA was analyzed following reversetranscription (RT) by Taqman PCR for the presence of ICP4 andglycoprotein C (gC) transcripts. DNA and RNA quantities were normalizedrelative to the cellular gene APRT.

Results are shown in FIG. 6. FIG. 6A shows that replication in the feetdepended both on heat treatment and small-molecule regulator.Furthermore, DNA replication was dependent on regulator dose. Compatibledata were obtained for the expression of ICP4 genes (regulated genes)and the late gC gene (not subject to deliberate regulation) (FIGS. 6B &C). Small-molecule regulator alone (in the absence of a heat treatment)did essentially not stimulate DNA replication and at most marginallyincreased expression of ICP4 and gC transcripts.

Replicative yields of HSV-GS3 and replication-defective virus KD6(ICP4-) were compared in the experiment shown in FIG. 6D. Thisexperiment was performed as the previous experiment with the exceptionthat mice were sacrificed 24 hours post heat induction. Results showedthat viral DNA could essentially only be detected in samples from thefeet of HSV-GS3-infected animals that had received heat treatment andulipristal. Very little DNA was found in DRG, and essentially none inKD-6-infected animals or in not-activated HSV-GS3-infected animals.

Example 5: Immunization/Challenge Experiment Comparing aReplication-Competent Controlled Virus of the Invention with aReplication-Defective Comparison Virus (KD6)

The goal of this type of experiment was to demonstrate that immunizingmice with the HSV-GS3 virus under inducing conditions elicits a strongprotective immune response against subsequent challenge with a lethaldose of wild-type HSV-1.

(a) Survival

Experimental Design:

TABLE 6 Immunization treatment groups HSV-GS3 Group Mock KD6 HSV-GS3(induced) Immunization vehicle 50,000 pfu of 50,000 pfu of 50,000 pfu ofTx* (MEM + 10% FBS) KD6 (non- HSV-GS3 HSV-GS3 replicating, ICP4-negativeHSV-1) Challenge Tx 10,000 pfu of 10,000 pfu of 10,000 pfu of 10,000 pfuof HSV-1 strain HSV-1 strain HSV-1 strain HSV-1 strain 17syn+ 17syn+17syn+ 17syn+ *All Tx were in a volume of 0.050 ml/mouse.a) Immunization:

Mice were initially immunized in the experimental groups shown in Table6. Each group contained 20 mice (ND4 Swiss Webster females, 4-6 weeks).Each vector was applied to the lightly abraded plantar surface of bothrear feet following saline pre-treatment.

b) Induction:

The HSV-GS3 vector was induced in the “HSV-GS3 (induced)” group asfollows: mifepristone (0.5 mg/kg) was administered i.p. at the time ofimmunization and again 24 h later. Heat was applied 3 h postimmunization by immersing both rear feet in a 43.5° C. water bath for 30min. Following immersion, the hind limbs were dried off, and the micekept warm with a heat lamp until dry and warm.

c) Challenge:

22 days post immunization the mice were challenged with a 20-fold lethaldose of wild type HSV-1 strain 17syn+ applied to the lightly abradedplantar surface of both rear feet following saline pre-treatment.Efficacy of each immunization treatment was then assessed by a modifiedendpoint assay. Note that the modified endpoint assay involvedeuthanizing mice that were considered moribund (will not survive) basedon clinical assessment (mice showing signs of bilateral hindlimbparalysis and CNS involvement, convulsions, and unable to move on theirown to take food or water).

Results:

Mice in the mock treatment group began to show signs of hindlimbparalysis and CNS infection as early as 6 days post challenge, while allthree immunization groups appeared completely healthy until 8-9 dayspost challenge. Table 7 depicts the number of mice surviving at the endof the experiment (mice were followed 30 d post challenge). The resultsdemonstrate that, while all treatments were able to protect at leastsome mice against a 20 fold lethal challenge of HSV-1, only the inducedHSV-GS3 virus treatment was able to afford substantial protection in themice.

TABLE 7 Results of immunization/challenge experiment No. of survivors(from groups of 20 Group animals) HSV-GS3 Induced 14 KD6 5 HSV-GS3 3Mock 0

A further experiment is presented below.

Experimental Design

a) Immunization:

Mice were initially immunized in the experimental groups shown in Table6. Each group contained 10 mice (ND4 Swiss Webster females, 4-6 weeks).Each vector was applied to the lightly abraded plantar surface of bothrear feet following saline pre-treatment.

b) Induction:

The HSV-GS3 vector was induced in the “HSV-GS3 (induced 2 times)” groupas follows: ulipristal (50 μg/kg) was administered i.p. at the time ofimmunization. Heat was applied 3 h post immunization by immersing bothrear feet in a 45° C. water bath for 10 min. Following immersion, thehind limbs were dried off, and the mice kept warm with a heat lamp untildry and warm. The latter activation procedure was repeated 24 h later.

c) Challenge:

approximately three weeks post immunization the mice were challengedwith a 2-fold lethal dose of wild type HSV-1 strain 17syn+ applied tothe lightly abraded plantar surface of both rear feet following salinepre-treatment. Efficacy of each immunization treatment was then assessedby a modified endpoint assay. Note that the modified endpoint assayinvolved euthanizing mice that were considered moribund (will notsurvive) based on clinical assessment (mice showing signs of bilateralhindlimb paralysis and CNS involvement, convulsions, and unable to moveon their own to take food or water).

TABLE 8 Immunization treatment groups HSV-GS3 induced Group Mock KD6HSV-GS3 2 times Vaccination Tx* vehicle 50,000 pfu of 50,000 pfu of50,000 pfu of HSV- (MEM + KD6 (non- HSV-GS3 GS3 10% FBS) replicatingICP4-HSV-1) Challenge Tx 1,000 pfu 1,000 pfu of 1,000 pfu of 1,000 pfuof HSV-1 of HSV-1 HSV-1 strain HSV-1 strain strain 17syn+ strain 17syn+17syn+ 17syn+ *All Tx were in a volume of 0.050 ml/mouse.Results:

Table 9 depicts the number of mice surviving at the end of theexperiment (mice were followed 30 d post challenge). The resultsdemonstrate that, while all treatments were able to protect at leastsome mice against a 2 fold lethal challenge of HSV-1, only the inducedHSV-GS3 virus treatment was able to afford substantial protection in themice.

TABLE 9 Results of immunization/challenge experiment No. of survivorsGroup (from groups of 10 animals) HSV-GS3 Induced 2 times 8 KD6 4HSV-GS3 3 Mock 2(b) Replication of Challenge VirusExperimental Design:a) Immunization:

Mice were immunized as shown in Table 6. Each group contained 5 mice(ND4 Swiss females, 4-6 weeks). Each vector was applied to the lightlyabraded plantar surface of the both rear feet following salinepre-treatment.

b) Induction:

The HSV-GS3 vector was induced in the “HSV-GS3 (induced)” group asfollows: mifepristone (0.5 mg/kg) was administered i.p. at the time ofimmunization and again 24 h later. Heat was applied 3 h postimmunization by immersing both rear hind feet in a 43.5° C. waterbathfor 30 min. Following immersion, the hindlimbs were dried off, and themice kept warm with a heat lamp until dry and warm.

c) Challenge:

22 days post immunization the mice were challenged with a 20-fold lethaldose of wild type HSV-1 strain 17syn+ applied to the lightly abradedplantar surface of the both rear feet following saline pre-treatment.Four days post challenge the mice were euthanized and feet weredissected and homogenized. The tissue homogenates were then diluted andtitrated on rabbit skin cells (RS) for infectious (17syn+) virus.

Results:

Table 10 depicts the results of the titration data. These resultsillustrate that, while all of the immunization treatments were able toreduce replication to some extent (relative to mock) in the feetfollowing challenge with HSV-1, HSV-GS3 (induced) mice showed by far thelowest challenge virus titer at four days post challenge (about twoorders of magnitude lower than mock).

TABLE 10 Infectious virus (pfu) detected in the feet of mice, 4 d postchallenge Mock KD6 HSV-GS3 HSV-GS3 (induced) 5.5 × 10⁵ +/− 4.0 × 10⁴ +/−9.4 × 10⁴ +/− 6.2 × 10³ +/− 1.5 × 1.2 × 10⁴ 8.3 × 10² 3.1 × 10⁴ 10²

Example 6: Adenovirus Vectors that Co-Replicate withReplication-Competent Controlled Virus

(a) Adenovirus Having a Replication-Essential Gene that is Activated bythe Transactivator of a Replication-Competent Controlled Virus

rAd3 lacks nucleotides 28,130-30,820 encompassing E3. Nucleotide numbersrelating to the adenovirus type 5 genome are as defined in GI: 33694637.Davison et al. 2003. J. Gen. Virol. 84, 2895-2908. Further, it containsa complete E1 gene that is functionally linked to a GAL4 site-containingminimal promoter.

The simplified system for generating recombinant adenovirus developed byHe et al. (Proc. Natl. Acad. Sci. USA 95: 2509-2514 (1998)) is employedto construct rAd3. This system has been made available commercially byStratagene Corp. of La Jolla, Calif. A manual entitled “AdEasy™Adenoviral Vector System” (revision no. 060002) is distributed by theCompany to its customers and is also available at the Company's website.

Mutagenesis is carried out in transfer vector pShuttle (He et al.(1998)) distributed by Statagene Corporation. For the completenucleotide sequence of this plasmid see FIG. 7 of U.S. Pat. No.7,906,312. According to Stratagene's manual entitled “AdEasy™ AdenoviralVector System” (revision no. 060002), pShuttle contains the followingadenovirus sequence elements: left inverted terminal repeat,encapsidation signal (Ad 1-331), “right arm homology region”(3,534-5790), “left arm homology region” (34,931-35,935) and rightinverted terminal repeat.

The right arm homology region of pShuttle is replaced with Ad sequencesalso containing the beginning of the E1 region in addition to thehomology region. First, pShuttle DNA is digested with NotI and PmeI, andthe vector fragment gel-purified. Next, a fragment containing Ad496-5780 is obtained by PCR amplification from plasmid pXC1 (MicrobixCorporation, Toronto, ON). The nucleotide sequence of this plasmid ispresented, e.g., in FIG. 9 of U.S. Pat. No. 7,906,312. The latter PCRamplification is carried out using a forward primer (reading into the E1gene) containing a SbfI restriction site and a reverse primer containinga PmeI restriction site. The resulting PCR fragment is digested withSbf1 and PmeI. Furthermore, a DNA fragment encompassing nucleotides 4634to 323 of plasmid pGene/V5-His (Invitrogen Corporation of Carlsbad,Calif.) is PCR-amplified using an appropriate forward primer containinga NotI restriction site and a reverse primer containing a SbfIrestriction site. The PCR-amplified fragment is digested with NotI andSbf1. The latter two end-digested PCR fragments are ligated to thepShuttle vector fragment. A plasmid containing the latter threefragments as evidenced by restriction analysis is termedpShuttle-E1-GAL4.

To prepare recombinant Ad, pShuttle-E1-GAL4 DNA is linearized by PmeIdigestion and co-electroporated with pAdEasy-1 DNA (He et al. 1998) intoE. coli BJ5183 cells. BJ5183 cells (Stratagene catalog no. 200154) havethe cellular components necessary to carry out homologous recombinationbetween introduced viral sequences. Detailed methods for the generationof recombinant Ad plasmids and the subsequent production of recombinantAd viruses are discussed in He et al. (1998) and in Stratagene manual“AdEasy™ Adenoviral Vector System” (revision no. 060002). Briefly,recombinant Ad plasmids are characterized by restriction digestion.After preparation of a sufficient amount of DNA of a correctrecombinant, the DNA is digested with PacI to separate plasmid sequencesand inserted sequences comprising the Ad sequences. To producerecombinant Ad virus, the digested DNA is transfected into 293 cells.Using standard technology (briefly described in He et al. (1998); seealso Graham and Prevec. 1991. Manipulation of adenovirus vectors. In:Methods in Molecular Biology, Gene Transfer and Expression Protocols,vol. 7, ed. Murray, The Humana Press Inc., Clifton, N.J., pp. 109-127),virus plaques are isolated, and rAd3 stocks prepared. Viral stocks canbe purified by CsCl gradient centrifugation or chromatographicprocedures.

In mammalian cells such as human fibroblasts co-infected with rAd3 andHSV-GS3 viruses, administration of an appropriate heat dose in thepresence of an effective concentration of small molecule regulator,e.g., mifepristone or ulipristal, results in activation of replicationof both viruses.

(b) Adenovirus Lacking a Replication-Essential Gene Complemented by aHSV-GS Virus that Conditionally Expresses the Ad Replication-EssentialGene

An E1A-defective Ad5 (rAd4) is prepared as follows: the right armhomology region of pShuttle is replaced with a longer Ad E1 sequence.First, pShuttle DNA is digested with NotI and PmeI, and the vectorfragment gel-purified. Next, a fragment containing Ad 496-5780 isobtained by PCR amplification from plasmid pXC1 (Microbix Corporation,Toronto, ON). The latter PCR amplification is carried out using aforward primer (reading into the E1 gene) containing a NotI restrictionsite and a reverse primer containing a PmeI restriction site. Theresulting PCR fragment is digested with NotI and PmeI. The latterend-digested PCR fragment is ligated to the pShuttle vector fragment. Aplasmid containing the latter three fragments as evidenced byrestriction analysis is termed pShuttle-E1.

To prepare recombinant Ad, pShuttle-E1 DNA is linearized by PmeIdigestion and co-electroporated with pAdEasy-1 into E. coli BJ5183cells. Recombinant Ad plasmids are characterized by restrictiondigestion. After preparation of a sufficient amount of DNA of a correctrecombinant, the DNA is digested with PacI to separate plasmid sequencesand inserted sequences comprising the Ad sequences. To producerecombinant Ad, the digested DNA is transfected into 293 cells. Usingstandard technology, virus plaques are isolated, and rAd4 stocksprepared. Viral stocks can be purified by CsCl gradient centrifugationor chromatographic procedures.

To prepare HSV-GS7, the GAL4 promoter-E1 region from plasmidpShuttle-E1-GAL4 is PCR-amplified using two of the same primers thathave been used in the construction of the latter plasmid: as forwardprimer is used the same forward primer that has been employed to amplifythe pGene/V5-His promoter fragment, and as reverse primer is used thesame reverse primer that has been employed to amplify the E1 fragmentfrom pXC1. (The primers are phosphorylated.) The PCR fragment isgel-purified. For the vector, pBS-KS:UL37/38 is digested with BspE1 andAfIII, and the resulting 3,772 bp fragment is treated with T4 DNApolymerase, gel-purified and SAP-treated, and then ligated to the abovePCR fragment. Following transformation, colonies are screened byrestriction digestion, and one colony is expanded that contains aplasmid with the correct insertion as verified by restriction enzymeanalysis and then by sequence analysis. This plasmid is termedpUL37/38:GAL4-E1.

One μg of pUL37/38:GAL4-E1 is co-transfected with 10 μg of purifiedHSV-GS3 virion DNA into E5 cells by calcium phosphate precipitation.Subsequent to the addition of mifepristone to the medium, thetransfected cells are exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently on days 2 and 3, the cells are againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaquesare picked and amplified on 96 well plates of E5 cells in mediasupplemented with mifepristone. The plates are incubated at 43.5° C. for30 minutes 1 hour after infection and then incubated at 37° C.Subsequently on days 2 and 3, the plates are also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells showed90-100% CPE, the plates are dot-blotted and the dot-blot membranehybridized with a ³²P-labeled oligonucleotide probe to the adenovirus E1region. A positive well is re-plaqued and re-probed several times andverified by sequence analysis to contain the expected GAL4-Ad5 E1 genesequence. This recombinant is designated HSV-GS7.

In mammalian cells such as human fibroblasts co-infected with rAd4 andHSV-GS7 viruses, administration of an appropriate heat dose in thepresence of an effective concentration of small molecule regulator,e.g., mifepristone or ulipristal, results in activation of replicationof both viruses.

All references cited in this application, including publications,patents and patent applications, shall be considered as having beenincorporated in their entirety.

The invention claimed is:
 1. A method of immunizing a mammalian subjectcomprising (a) administering to an inoculation site region in the bodyof a mammalian subject a composition comprising an effective amount of areplication-competent controlled herpesvirus which is a recombinantvirus in which one or more replication-essential genes have been placedunder the control of a gene switch that is inserted in the genome of therecombinant virus and that can be activated deliberately, and (b)exposing the inoculation site region of the mammalian subject to alocalized activation treatment that activates the recombinant virus toundergo a round of replication in the inoculation site region andinduces a functional immune response, wherein the inoculation siteregion is a cutaneous region, a subcutaneous region, or a mucosalmembrane and the localized activation treatment comprises a heattreatment.
 2. The method according to claim 1, wherein thereplication-competent controlled herpesvirus is a recombinant virusselected from the group consisting of an HSV-1, an HSV-2, a varicellazoster virus, a cytomegalovirus and a roseola virus.
 3. The methodaccording to claim 1, wherein the replication-competent controlledherpesvirus is a recombinant herpesvirus that comprises an inserted geneencoding a small-molecule regulator-activated transactivator which geneis functionally linked to a nucleic acid sequence that acts as a heatshock promoter or to a nucleic acid sequence that acts as a heat shockpromoter as well as a transactivator-responsive promoter, and one ormore transactivator-responsive promoters that are functionally linked tothe one or more replication-essential genes.
 4. The method according toclaim 3, wherein the replication-competent controlled herpesvirusfurther comprises at least one gene from another pathogen, aheterologous gene encoding an immune-modulatory polypeptide and aheterologous gene encoding another polypeptide.
 5. The method accordingto claim 1, wherein the replication-competent controlled herpesviruscomprises a gene that encodes an HIV envelope protein antigen or an HIVinternal protein antigen.
 6. The method according to claim 3, whereinthe replication-competent controlled herpesvirus is a recombinant virusselected from the group consisting of an HSV-1, an HSV-2, a varicellazoster virus, a cytomegalovirus and a roseola virus.
 7. The methodaccording to claim 3, wherein the replication-competent controlledherpesvirus is a recombinant HSV-1 or HSV-2 and thereplication-essential viral genes that are functionally linked totransactivator-responsive promoters include at least all copies of theICP4 gene or the ICP8 gene.
 8. The method according to claim 3, whereinthe small-molecule regulator-activated transactivator contains atruncated ligand-binding domain from a progesterone receptor and isactivated by a progesterone receptor antagonist or other moleculecapable of interacting with the ligand-binding domain and activating thetransactivator.
 9. The method according to claim 1, wherein thereplication-competent controlled herpesvirus is a recombinant HSV-1selected from the group consisting of HSV-GS1, HSV-GS3, HSV-GS4 andHSV-GS6, wherein the recombinant HSV-1 further comprises at least onegene from another pathogen, a heterologous gene encoding animmune-modulatory polypeptide and a heterologous gene encoding anotherpolypeptide.
 10. The method according to claim 3, further comprisingadministering a second virus that has a host range that overlaps that ofthe replication-competent controlled herpesvirus, the second virushaving a replication-essential gene functionally linked to atransactivator-responsive promoter.
 11. The method according to claim 3,further comprising a second virus that has a host range that overlapsthat of the replication-competent controlled herpesvirus, wherein areplication-essential gene from the second virus is expressed under thecontrol of a transactivator-responsive promoter by thereplication-competent controlled herpesvirus and the second virus isdefective in said replication-essential gene.
 12. The method accordingto claim 3, wherein the composition that is administered to theinoculation site region further comprises an effective amount of asmall-molecule regulator that is capable of activating thetransactivator.
 13. The method according to claim 3, wherein: (a) thecomposition comprising an effective amount of the replication-competentcontrolled herpesvirus is administered to the inoculation site region,and (b) the inoculation site region is exposed to an activating heatdose in the presence in the inoculation site region of an effectiveconcentration of a small-molecule regulator that activates thetransactivator of the replication-competent controlled herpesvirus. 14.The method according to claim 12, wherein: (a) the composition furthercomprising an effective amount of a small-molecule regulator isadministered to the inoculation site region, and (b) the inoculationsite region is exposed to an activating heat dose.
 15. The methodaccording to claim 1, wherein the replication-competent controlledherpesvirus is a recombinant herpesvirus that comprises an inserted geneencoding a transactivator activated by a small-molecule regulator,wherein the gene encoding the transactivator is functionally linked to anucleic acid that acts as a heat shock promoter, and one or moretransactivator-responsive promoters that are functionally linked to oneor more replication-essential genes.
 16. The method according to claim15, wherein the replication-competent controlled herpesvirus furthercomprises at least one gene from another pathogen, a heterologous geneencoding an immune-modulatory polypeptide and a heterologous geneencoding another polypeptide.
 17. The method according to claim 1,wherein the replication-competent controlled herpesvirus is arecombinant herpesvirus that comprises an inserted gene encoding asmall-molecule regulator-activated transactivator wherein the geneencoding the transactivator is functionally linked to a nucleic acidthat acts as a constitutively active promoter or atransactivator-responsive promoter, a first replication-essential geneof the replication-competent controlled herpesvirus is functionallylinked to a promoter activated by heat and a secondreplication-essential gene of the replication-competent controlledherpesvirus is functionally linked to a transactivator-responsivepromoter.
 18. The method according to claim 17, wherein thereplication-competent controlled herpesvirus further comprises at leastone gene from another pathogen, a heterologous gene encoding animmune-modulatory polypeptide and a heterologous gene encoding anotherpolypeptide.
 19. The method according to claim 1, wherein thereplication-competent controlled herpesvirus is a recombinant HSV-1selected from the group consisting of HSV-GS1, HSV-GS3, HSV-GS4 andHSV-GS6.